Jan 14, 1988 - milton PRP-1; 250 by 4.1 mm), with a flow rate of 1 ml/min with a two-buffer (buffer A, ..... Durham, D. R., C. G. McNamee, and D. B. Stewart. 1984. ... phenylalanine ammonia-lyase of Rhodosporidium toruloides. J. Bacteriol.
APPLIED
AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1988, 0099-2240/88/040996-07$02.00/0 Copyright © 1988, American Society for Microbiology
p. 996-1002
Vol. 54, No. 4
Strain Improvement of Rhodotorula graminis for Production of a Novel L-Phenylalanine Ammonia-Lyase STEVE A. ORNDORFF,t* NINA COSTANTINO,t DAVID STEWART, AND DON R. DURHAM§ Protein Engineering Department, Genex Corporation, Gaithersburg, Maryland 20877 Received 19 October 1987/Accepted 14 January 1988 L-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Rhodotorula rubra has been used in the commercial manufacture of L-phenylalanine from trans-cinnamic acid. In this study, R. graminis PAL was investigated. Mutant strain GX6000 was isolated after ethyl methanesulfonate mutagenesis of wild-type R. graminis GX5007 by selecting for resistance to phenylpropiolic acid, an analog of trans-cinnamic acid. Mutant strain GX6000 produced inducible PAL at levels four- to fivefold higher than had wild-type R. graminis. Furthermore, this strain had several other physiological traits that make it more commercially useful than R. rubra. For example, during fermentation, the PAL half-life was three- to fivefold longer, PAL specific activity was six to seven times higher, and PAL synthesis was significantly less inhibited by temperatures above 30°C. Induction of PAL in strain GX6000 appeared to be less tightly regulated; L-leucine acted synergistically with L-phenylalanine, the physiological inducer, to increase the PAL specific activity and titer to 165 U/g (dry weight) and 3,000 U/liter, respectively, a 40% increase over the effect of L-phenylalanine alone. Strain GX6000 PAL showed significantly greater stability in bioreactors for the synthesis of L-phenylalanine, a finding that is consistent with the stability properties observed during fermentation.
L-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyzes the deamination of L-phenylalanine to trans-cinnamic acid and ammonia in a variety of plants and microorganisms. It is ubiquitous in plants as the initial enzyme in biosynthetic pathways for the formation of many phenylpropanoid compounds, including lignin, flavonoids, and other phenolic derivatives (17). In microorganisms, PAL functions as a catabolic enzyme, allowing yeasts, fungi, and streptomycetes to utilize phenylalanine as a sole source of carbon and energy or as a sole nitrogen source (6, 16). PAL has various industrial and potential medical applications. Purified PAL is suitable for the treatment of certain mouse neoplastic tumors (5), the quantitative analysis of serum phenylalanine (13), and the treatment of phenylketonuria (10). It has also been used commercially in the manufacture of L-phenylalanine by reversing the enzyme reaction with high concentrations of trans-cinnamic acid and ammonia at an elevated pH (7). Earlier attempts at the biosynthesis of L-phenylalanine from trans-cinnamic acid were of limited success, partly because of the relatively low specific activity and rapid turnover of PAL during microbial fermentations (19). PAL isolated from yeasts, particularly Rhodotorula spp., has been the principal source of the enzyme for commercial applications. Therefore, it was of interest to develop a more productive yeast fermentation. In this report, we describe a natural isolate of a Rhodotorula strain, Rhodotorula graminis, that has several desirable properties as a production strain. For example, PAL from R. graminis exhibits exceptional stability during fermentation, and this species has nonfastidious requirements for growth and PAL synthesis. However, the microorganism synthe-
sizes low levels of PAL during fermentation. Mutants that express PAL at high levels were selected as phenylpropiolic acid (PPA)-resistant isolates. PAL production and regulation by one of these mutants, strain GX6000, is described here and compared with those by other production strains (7). Interestingly, induction of R. graminis PAL was affected synergistically by the combination of L-phenylalanine and L-leucine. MATERIALS AND METHODS Strains and culture conditions. R. graminis KGX39 (GX5007) wild type was isolated from soil and used as the parental strain for isolating analog-resistant mutants. R. rubra GX3243 was as described elsewhere (J. C. McGuire and H.-H. Yang, U.S. patent 4,636,466, January 1987). A detailed description of strain GX5007 was reported by Durham et al. (1). Working stock cultures were maintained on YPD agar medium (3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 10 g of glucose, and 5 g of L-phenylalanine per liter; pH 6.0). Diauxic growth studies were done in shake flasks containing basal salts medium (BSM; 2), 0.5% yeast extract, 5 mM glucose, and 10 mM L-phenylalanine (pH 7.0). Cultures were incubated at 30°C with agitation (250 rpm). Standard PAL fermentations were performed in 2- or 10-liter fermentors by using a fed-batch operation and working volumes of 1.5 and 7.5 liters, respectively. PAL fermentation medium was composed of 5 g of yeast extract, 2 g of (NH4)2P04, 14 g of glucose, and 6 g of L-phenylalanine per liter. Successive feeds of 50% glucose (14 g/liter, final concentration in fermentor) and 30% yeast extract (15 g/liter, final concentration in fermentor) were added after exhaustion of the initial glucose during growth. Fermentor operating conditions were as follows: 800 rpm, 1.0 volume of air per volume (medium) per min (vol/vol/min), pH 6.0 + 0.1, 30°C, 10% (vol/vol) inoculum. Control of pH was maintained by automatic addition of 6 N NaOH or 6 N H2SO4. Mutant isolation. R. graminis GX5007 was grown overnight at 30°C in BSM containing 20 mM glucose. A 5-ml portion of cells was used to inoculate fresh medium, and
* Corresponding author. t Present address: The NutraSweet Co., Box 2387, Augusta, GA 30903. t Present address: National Cancer Institute, Frederick Cancer
Research Facility, Frederick, MD 21701. § Present address: W. R. Grace & Co., Washington Research Center, Columbia, MD 21044.
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L-PHENYLALANINE AMMONIA-LYASE FROM R. GRAMINIS
growth was continued for an additional 4 h. Equal portions of washed cells were transferred into eight tubes and mutagenized with 3% (vol/vol) ethyl methanesulfonate for 30 to 60 min, which was sufficient for a greater than 90% killing of the cells. The cells were harvested, washed three times, and inoculated into BSM-glucose medium for recovery of viable cells. Overnight cell suspensions were spread onto BSMagar medium containing 10 mM L-phenylalanine and either 10 or 50 p.M PPA. Mutants appearing after 3 to 4 days were purified onto BSM-agar medium containing 10 mM L-phenylalanine and 100 F.M PPA. PPA-resistant mutants were screened for elevated PAL specific activity in shake flasks and further screened with 2-liter fermentations. PAL assays. PAL activity was determined by a modification of the procedure of Kalghatgi and Subba Rao (11) with permeabilized viable-cell suspensions. The standard reaction mixture contained 25 mM L-phenylalanine, 25 mM Tris buffer (pH 8.8), 0.001 to 0.005% (wt/vol) cetylpyridinium chloride, and an appropriate volume of cells. The rate of formation of trans-cinnamic acid at 30°C was determined by measuring the increase in A280 with a Gilford 260 recording spectrophotometer. One unit of PAL activity was defined as the amount of enzyme required to convert one micromole of L-phenylalanine to trans-cinnamic acid per minute. PAL activity is expressed as units of enzyme per liter (titer) or units per gram (dry weight) of cells (specific activity). PAL specific activity in the diauxic growth studies was calculated as the rate of increase in absorbance per minute per cell density at 600 nm (A28Jmin - optical density [OD]). PAL inducer specificity studies. Induction of PAL activity was determined by a modification of the method of Nakamichi et al. (15). Cells were grown overnight in 100 ml of YPD broth medium without L-phenylalanine, harvested by centrifugation, washed, and suspended in 10 ml of 50 mM potassium phosphate buffer (pH 6.0). An induction medium containing 1.0 ml of cell suspension, 50 mM potassium phosphate buffer (pH 7.0), and inducer in a final volume of 25 ml was incubated at 30°C with agitation (250 rpm). Inducer concentrations are noted in the tables and figures. PAL activity was measured for 10 to 12 h. Bioreactor experiments. L-Phenylalanine was synthesized from trans-cinnamic acid and ammonia in 1-liter anaerobic bioreactors containing Rhodotorula cells that were harvested from 2-liter standard PAL fermentations at peak PAL activity. Bioreactor medium contained 15 g of trans-cinnamic acid, 120 g of (NH4)2CO3, 350 ml of NH40H (29%), and sufficient cell paste to yield 800 to 1,000 U of PAL in a final volume of 1 liter (pH 10.5). The bioreactor was sparged with nitrogen and incubated at 30°C for about 100 h. LPhenylalanine and trans-cinnamic acid concentrations were determined as described below and used as the basis for the addition of trans-cinnamic acid to the bioreactors during incubation. After the bioreaction, the cells were harvested by centrifugation, washed, and assayed for PAL activity. Analytical methods. L-Leucine and L-phenylalanine concentrations in fermentation samples were assayed by highpressure liquid chromatography analysis of o-phthalaldehyde-derivatized samples. Pre-column derivatization of the fermentation supernatant samples with o-phthalaldehyde was followed immediately by reversed-phase high-pressure liquid chromatographic separation on a C18 column (Hamilton PRP-1; 250 by 4.1 mm), with a flow rate of 1 ml/min with a two-buffer (buffer A, acetate-methanol-p-dioxane-tetrahydrofuran [THF]; buffer B, methanol-THF) gradient (Waters Corp., Milford, Mass.), excitation at 330 nm, and detection at 418 nm. L-Phenylalanine and L-leucine
997
eluted at 34 and 37 min, respectively. trans-Cinnamic acid concentrations in the bioreactor were measured at 268 nm with diluted samples of bioreactor supernatant and then calculated on the basis of its extinction coefficient (E = 10,000/M per cm). Glucose was assayed with a YSI model 27 Industrial Analyzer (Yellow Springs Instrument Co., Yellow Springs, Ohio). Chemicals. Amino acids were purchased from Sigma Chemical Co. (St. Louis, Mo.). Technical-grade trans-cinnamic acid was purchased from Eastman Kodak Co. (Rochester, N.Y.). Amberex 1003 yeast extract was purchased from Universal Foods Corporation (Milwaukee, Wis.). All other reagents and chemicals were of analytical grade. RESULTS Fermentations of R. graminis for PAL formation. Fermentations of R. graminis GX5007 were performed in 2-liter fermentors with PAL production medium that contained L-phenylalanine (6 g/liter) as the inducer. The fermentations received a glucose feed after initial glucose exhaustion, followed by a yeast extract feed after exhaustion of the glucose supplement (Fig. 1). PAL activity was detected only after carbohydrate disappearance, indicating glucose-catabolite repression (Fig. 1). R. graminis fermentations yielded a volumetric titer of 450 U/liter and a specific activity of 26 U/g of cells. By comparison, parallel fermentations with R. rubra GX3243 exhibited a titer of 1,204 U/liter and a specific activity of 77 U/g of cells. The specific productivity values (units per gram of cells per gram of L-Phe), which is a measure of inducer efficiency, were 4.3 and 12.8 for R. graminis and R. rubra, respectively. Despite the low titers of PAL observed during the fermentation of R. graminis, a noteworthy feature of the fermentation was the marked stability of PAL (Fig. 1). For example, PAL from R. graminis exhibited a half-life during fermentation of 8 h as compared with 3 h for the R. rubra enzyme. Isolation of PPA-resistant mutants of R. graminis. To increase the PAL titer in R. graminis, mutants were isolated after chemical mutagenesis and selected for their ability to utilize L-phenylalanine as the sole carbon and energy source in the presence of PPA. At low levels (10 ,uM) PPA inhibits the growth of wild-type R. graminis on BSM containing L-phenylalanine, but not on medium containing cinnamate or glucose. In addition, PPA inhibits the deamination of phenylalanine to trans-cinnamic acid in vitro (9, 17). Approximately 500 mutants were isolated that grew on phenylalanine in the presence of PPA. Colonies appearing after 3 days were purified on phenylalanine minimal medium containing 100 ,LM PPA.
Screening of PPA-resistant mutants for elevated levels of PAL. PPA-resistant mutants were initially screened by measuring PAL specific activity during cell growth in shake flasks containing modified BSM, 5 mM glucose, and 10 mM L-phenylalanine. Glucose was included as a cosubstrate to permit cells to reach a uniform cell density and hence a similar physiological state before induction of PAL. Under these conditions, R. graminis exhibits diauxic growth (1), initially utilizing glucose and then L-phenylalanine; PAL induction is apparent after glucose utilization (Fig. 2). Eight independently isolated mutants had significantly higher PAL activities as compared with those of the parental strain. A comparison of one mutant strain, GX6000, to wild-type R. graminis is shown in Fig. 2. R. graminis GX6000 had a slightly faster doubling time on L-phenylala-
APPL. ENVIRON. MICROBIOL.
ORNDORFF ET AL.
998
I
16
f
i
la =
l a
a5-
a
C
4
S.
o 0.
I0ft L) a
C
4c
92
.
6
ii
I c
e
aC
I
E0Ud
4
I
i
I
-2
Eco
I
A. 0
8
I.
0
I
0
0
30
20
10
Hours
Hours
FIG. 1. Comparison of Rhodotorula spp. during standard PAL fermentation. Fermentations were carried out as 2-liter fed-batch fermentations in PAL medium with successive feeds (at times indicated by arrows) of glucose (14 g/liter) and yeast extract (15 g/liter). Operating conditions are described in Materials and Methods.
nine and expressed PAL at threefold-higher levels under these conditions. This mutant, as well as all of the others tested, did not produce PAL constitutively or in the presence of glucose (Fig. 2) and exhibited a decay in PAL activity after reaching peak levels at approximately 10 to 12 h. Mutants were evaluated further in 2-liter fermentors with PAL production medium. All PPA-resistant mutants, except .3
430 R. graminis GX5007
(wild-type)
380 330_ 280 230
.25,
430 R. graminis GX6000 360 330280
.230
Cell Growth
180
one, from the primary screening produced two- to threefoldhigher PAL titers and specific activities than did the wildtype R. graminis (Table 1). Similarly, PAL specific activity was increased in these strains. Characteristics of R. graminis GX6000 fermentations. A comparison of various fermentation parameters for R. graminis GX6000 and the parental strain, in 2-liter fermentations, is shown in Table 2. Strain GX6000 consistently
(.25
-
Cell Growth .2
.2
fth
130-
i ,
.1. 130O< 80 '/d|
2
2130
-.15 14 10-~~~~~~~~~~-
d~~~~~~~~~~~~~~~~~a
c
0
.3 PAL
312 0
1
2
3
6 4-
4
5
Hours
6
--O78
7
8
913
9 10
j0
1
2
3
4
5
6
7
8
9
Hours
FIG. 2. Induction of PAL in R. graminis wild type (GX5007) and PPA-resistant mutant (GX6000). Strains were inoculated into shake flasks containing BSM with either 0.5% yeast extract and 5 mM glucose (dashed line) or 5 mM glucose and 10 mM L-phenylalanine (solid line) and then incubated at 30°C with 250-rpm shaking.
TABLE 1. Evaluation of PAL productivity in PPA-resistant mutants of R. graminis GX5007 in 2-liter fermentations' PAL productivity R. graminis
Sp
Titer
act
(U/g of cells)
(U/liter)
Specific productivity (U/g of cells per g of L-Phe)
26
414
4.4
Mutants GX6000 PP-1 PP-7 PP-501 PP-502 PP-503 PP-504 PP-801
79 24 52 74 52 58 45 58
1,300 426 833 1,204 917 972 749 1,020
13.2 4.0 8.7 12.5 8.7 9.7 7.5 9.8
exhibited PAL titers and specific activities that were fourfold greater than those for R. graminis wild type. The degree of
PAL stability, as measured by half-life duration, was slightly greater in GX6000 than in the wild-type strain. All other parameters examined, e.g., growth and PAL induction ki-
netics, were similar (Table 2). (i) Effect of phenylalanine on PAL induction. PAL titers during fermentation increased in proportion to the L-phenylalanine concentrations to 9 glliter. Higher concentrations of phenylalanine gave negligible increases in PAL titer or specific activity. In addition, D-phenylalanine and DL-phenylalanine were effective inducers of PAL (data not shown). (ii) Effect of various amino acids on PAL induction. To obtain less expensive inducers, the effect of individual alternative amino acids and several combinations of amino acids on PAL induction was assessed in shake flask studies. The data demonstrate that various amino acids, in particular hydrophobic amino acids, induce PAL to levels equivalent to or greater than those observed with the physiological inducer L-phenylalanine (Table 3). The combination of Lphenylalanine and L-leucine increased PAL activity in the mutant to higher levels than those observed with the individual amino acids. The effect of various isomeric combinations of phenylalanine and leucine on PAL activity was determined in R. graminis GX6000 since the L-isomer combination of these amino acids had induced the highest PAL specific activity. Shake flask tests indicated that L-leucine in combination with any phenylalanine isomer induced the highest PAL TABLE 2. Comparison of R. graminis wild-type and mutant PAL fermentationsa PAL
Maximum Specific
Titer
(U/g) (U/liter)
GX5007 (wild type) GX6000 (mutant)
26 105
410
1,680
Peak PAL sp act (U/g of
cells)b
15.8 31.6 26.7 19.4 10.9 Tyrosine ......... 1.2 Tryptophan ......... Valine ......... 23.1 Phenylalanine + Leucine ......... 34.0 + Isoleucine ......... 21.9 + Valine ......... 13.4
Phenylalanine ......... Isoleucine ......... Leucine ......... Methionine .........
a Two-liter fed-batch fermentations in PAL medium with 6 g of L-phenylalanine inducer per liter.
Sp act
TABLE 3. Effect of various amino acids on PAL induction in R. graminis GX6000 Inducer'
Wild type (GX5007)
Strain'
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L-PHENYLALANINE AMMONIA-LYASE FROM R. GRAMINIS
VOL. 54, 1988
a L-Amino acids: single-inducer concentration, 6 g/liter; double-inducer concentrations, L-phenylalanine and second amino acid, 6 and 1 g/liter, respectively. Other amino acids that were tested and failed to induce significant PAL activity were L-Ala, L-Arg, L-Asp, L-Cys, L-Glu, L-His, L-Lys, L-Ser, and L-Thr. b Cells were grown in YPD broth in shake flasks, and induction was carried out with washed cells as described in Materials and Methods. PAL activity was measured hourly for 10 h.
specific activity in R. graminis GX6000 (Table 4). The level of this combined action was related to the phenylalanine isomer used with leucine; the highest PAL specific activity was found with the DL-isomeric mixture, followed by the L-isomer alone and then by the D-isomer alone. This pattern of induction was also observed when only the phenylalanine isomers were used as the inducer. In contrast, D-phenylalanine failed to induce significant PAL activity in R. rubra strains (data not shown). The augmentation of PAL specific activity by L-leucine addition to standard PAL fermentation medium was analyzed in 2-liter fermentations. These results are summarized in Fig. 3. PAL activity increased in proportion to the total concentration of inducer or increasing quantity of either amino acid, indicating that the combination of L-phenylalanine and L-leucine had a synergistic inducer effect. The maximal PAL titer and specific activity values with leucine supplementation (1 g of L-leucine and 6 g of L-phenylalanine per liter) were 2,977 U/liter and 153 U/g, respectively, in 10-liter fed-batch fermentations. TABLE 4. Effect of various leucine and phenylalanine isomer combinations on induction of PAL in R. graminis GX6000' Maximum PAL sp act (U/g)
Increased sp act due to leucinec
L-Leu + DL-Phe L-Leu + L-Phe L-Leu + D-Phe
74.5 63.1 63.1
31.9 37.3 45.9
DL-Leu + DL-Phe DL-Leu + L-Phe DL-Leu + D-Phe
60.2 57.3 47.3
17.6 31.5 30.1
DL-Phe L-Phe D-Phe DL-Leu L-Leu
42.6 25.8 17.2 25.8 24.4
Inducer
combinationb
cell
productivity Half-life
dry
wt
(U/g per g of L-Phe)
(h)c
(g/liter)
4.3 17.5
8 11
16.9 15.7
Two-liter fermentations with PAL fermentation medium containing 6 g of per liter. b Number of fermentation runs (n) was 4 and 17 for GX5007 and GX6000,
a
L-phenylalanine
respectively. c Time taken for 50% decay of maximum observed PAL activity.
a Cells were grown in YPD broth in shake flasks, and induction was carried out with washed cells. b Concentrations of combined inducers: leucine isomers, 1 g/liter; phenylalanine isomers, 6 g/liter; single amino acid inducer concentration, 6 g/liter. c Compared with that due to corresponding phenylalanine isomer.
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ORNDORFF ET AL.
APPL. ENVIRON. MICROBIOL.
of yeast on PAL fermentation. * GX6000 GX6O0(iii) R. graminis additionEffect of yeast extractextract during the induction phase (Fig.The 1) had no observable effect on PAL specific activity during fermentation of R. graminis GX6000. For example, without yeast extract, a specific activity of 102 was obtained as (3) compared with 111 for a parallel fermentation supplemented with yeast extract (data not shown). However, the omission / of yeast extract lengthened the period required to reach (6) maximal PAL activity, albeit by only 1 to 2 h. (1) Effect of temperature on induction of PAL. The effect of Constant L-phenylalanine, temperature on PAL synthesis, titer, and stability was
200
150 Constant L-leucine,
'0
variable Lphenyalanine
la
variable L-leucine
examined. Both R. graminis and R.
t(5S)
(4) 100 L / 00 (.25) 2w
|
I%.
L-phanylalanin.
A
50
2
4
6
8
10
12
Total Amino Acid Inducer (gm/I) FIG. 3. Synergistic effect of various L-phenylalanine/L-leucine Indlucer ratios on PAL productivity in R. graminis GX6000 in 2-liter fed[-batch fermentations in PAL medium. The concentration of the var^iable amino acid is given in narentheses. The concentrations of constant L-leucine and L-phenylalanine were 1 and 6 g/liter, respectively.
R.
rubra GX3243
rubra yielded maximum
activity when cultivated at 20 to 30°C in shake flask induction experiments and lower activity at temperatures greater than 30°C (Fig. 4). However, PAL activity in R. rubra was significantly lower than that in R. graminis at temperatures greater than 30°C. PAL activity in R. rubra was reduced approximately 75% at 35°C from the maximum activity found at 25°C, whereas R. graminis PAL activity was depressed only 30%. Under the conditions of this experiment, both strains demonstrated turnover of PAL that led to a loss of activity following peak activity. Bioreactor experiments with R. graminis. The stability of R. graminis PAL found during fermentations was also evident in the synthesis of L-phenylalanine from trans-cinnamic acid in bioreactors. PAL half-life and final PAL activity were nearly twofold greater than with R. rubra in concurrent experiments (Table 5). All other aspects of the bioreactor performance were equivalent between the two strains. However, it should be noted that bioreaction conditions were .'. optimzed for R. rubra and no attempt was made to adjust PAL
conditions specifically for R.
graminis.
DISCUSSION PAL mediates the stereospecific addition of ammonia to trans-cinnamic acid to form phenylalanine. This reaction has R.
graminis GX6000
0.5
O 0.4 0
E
0.2 330
Induction Time (Hours) FIG. 4. Effect of temperature on PAL synthesis in R. rubra GX3243 and R. graminis GX6000. Each strain was grown in BSM containing 1% yeast extract and 1% glucose for 15 h at 30°C in shake flasks. Washed cells were added to BSM (pH 7) and induced with 10 mM L-phenylalanine.
L-PHENYLALANINE AMMONIA-LYASE FROM R. GRAMINIS
VOL. 54, 1988
TABLE 5. Whole-cell bioconversion of trans-cinnamic acid to L-phenylalanine by R. rubra GX3243 and R. graminis GX6000' Strainb
R. rubra GX3243 R. graminis GX6000
PAL % Conversion Final PAL L-Phe of transhalf-life activity production (% of initial) (g/liter) cinnamic acid (h)
30 54
18 36
46.7 50.8
82 86
a One-liter anaerobic bioreactors incubated at 22°C for 88 h as described in Materials and Methods. Initial PAL activity in all bioreactors was approximately 800 U/liter. b Each strain tested in duplicate bioreactors.
been exploited in a commercial process with yeast of the genus Rhodotorula (7). We previously described the isolation of a yeast, R. gratninis, by enrichment culture with benzoate as a sole source of carbon and energy (1). Unlike many Rhodotorula species, this strain grows readily on aromatic substrates, and aromatic acids are not inhibitory (1). Furthermore, this isolate represents the only yeast strain that expresses benzoate-4-hydroxylase at levels that can be measured in vitro (1). On the basis of these observations, we predicted that R. graminis may contain high levels of PAL and thus could potentially serve as a suitable organism for the production of PAL biocatalyst for the synthesis of L-phenylalanine. Fermentations with R. graminis indicated that it produced only moderate levels of PAL, volumetric titers being 2- to 2.5-fold lower than those of R. rubra mutant production strains (Fig. 1). However, R. graminis grew 20% faster than R. rubra, seed preparation time was reduced by a factor of 2, and the time of fermentation required to reach the maximum PAL titer was 15 to 20 h compared with 23 to 30 h for R. rubra (data not shown). Most significantly, the PAL produced during aerobic fermentation of R. graminis was more stable than any reported source of the enzyme (4, 6, 11, 14). PAL from most microorganisms is readily turned over during cultivation (4, 6, 16, 19). Methods to reduce R. rubra PAL degradation include nitrogen sparging and cooling of the fermentation medium to 10 or 20°C during the induction phase (4; M. A. Finkelman et al., U.S. patent 4,584,273, April 1986) and the inclusion of alcohols (3) or alternative amino acids such as L-isoleucine (4, 16). The greater stability of R. graminis PAL obviates the need to use these stabilization methods (Fig. 1). However, mutant strains that produce higher volumetric titers of PAL had to be isolated to develop a commercially significant production strain. Analogs of substrates of catabolic pathways can be used to select mutants that overproduce enzymes of dissimilatory pathways (8). McGuire et al. (U.S. patent 4,636,466, January 1987) used various analogs of L-phenylalanine and several tyrosine isomers as the sole carbon source in minimal salts medium for the selection of PAL-overproducing microbial strains from mutagenized cells. R. graminis degrades Lphenylalanine through cinnamic acid and the P-ketoadipate pathway (1). Cinnamic acid and analogs thereof are competitive inhibitors of PAL (9, 17). PPA, an acetylenic analog of cinnamate, is a potent, noncompetitive inhibitor of PAL from yeast strains (9). Growth of R. graminis is inhibited by PPA during growth on phenylalanine, but not during growth on cinnamate or nonaromatic substrates. Mutants resistant to the inhibitory effects of PPA were isolated after chemical mutagenesis, and several of these mutants were selected for further evaluation and shown to express PAL at levels threeto fourfold higher than had wild-type R. graminis (Table 1). However, none of these mutants expressed PAL constitu-
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tively. The levels of PAL stability, the maximum cell (dry weight) yields, the doubling times, and the fermentation times required to reach maximal PAL production were similar among the mutants and the parental strain. Thus, the mutagenesis did not affect any structural or regulatory genes critical to cell metabolism and growth or the synthesis of PAL. The regulation of PAL synthesis is complex and occurs at the transcriptional level in yeast cells with L-phenylalanine functioning as the physiological inducer of the enzyme (6, 14, 19). In complex media, PAL activity in R. glutinis appears during stationary-phase growth, since catabolism of L-phenylalanine is required only under carbon- or nitrogenlimited conditions (14). In addition, glucose acts as a catabolite repressor (Fig. 1) (4, 6), whereas ammonia, in the presence of glucose, represses the induction of PAL synthesis by L-phenylalanine in a manner not fully understood (4, 6, 14). A report by Nakamichi et al. (15) suggested that other amino acids, in particular D- or L-isoleucine, D- or L-leucine, L-methionine, L-tryptophan, and L-tyrosine induced PAL activity in R. glutinis. These investigators proposed that these amino acids exerted their effect at the transcriptional level by an unknown mechanism by which the PAL repressor was inactivated. Similarly, PAL from R. graminis is induced by other amino acids, in particular, L-isoleucine, L-valine, and L-leucine (Table 4). The level of R. graminis GX6000 PAL was also comparatively high when D- or L-phenylalanine and the DL-isomeric mixture were used as an inducer. A similar observation has been made by Evans et al. (4) with four different species of Rhodotorula natural isolates, although Nakamichi et al. (15) observed lower PAL activity in R. glutinis with D-phenylalanine. It also has been found that fungi readily metabolize either isomer of phenylalanine via cinnamic acid or phenylpyruvic acid (12). The use of D-phenylalanine or the DL-isomeric mixture has significant implications for the economics of the PAL process for production of L-phenylalanine since the D-isomer can be chemically synthesized at a much lower cost than the L-isomer. The concentration of any individual inducer required for maximum PAL activity in the parental or mutant strains of R. graminis was 6 to 9 g/liter, although substantial activity was observed with 3 g of inducer per liter. Most Rhodotorula spp. appear to be fully induced when inducer is used in the concentration range of 5 to 10 g/liter (15), although Evans et al. (4) have described a mutant strain fully induced at a 2-g/liter concentration of L-phenylalanine. Such observations suggest that mutant PAL strains with significantly lower inducer requirements can be obtained. Our data suggest that combinations of L-leucine (1 g/liter) with isomers of phenylalanine (6 g/liter) result in increases of PAL specific activity of 32 to 46 U/g over that observed with phenylalanine isomers alone (Table 4). Furthermore, PAL specific activity can be increased by varying the molar ratios of L-phenylalanine to L-leucine (Fig. 3). In comparative 10-liter fermentations, the PAL titer and specific activity values were 1.6-fold higher when L-leucine (1 g/liter) was added to the PAL production medium. Thus, the combination of L-leucine and phenylalanine synergistically increases PAL specific activity and titer in R. graminis. Our results differ from those of Nakamichi et al. (15) and Yamada et al. (19), who reported that L-isoleucine was principally used to increase PAL stability rather than increase PAL titers in R. glutinis. Interestingly, L-isoleucine or L-leucine did not have a significant effect on extending the stability of the PAL from R. graminis (data not shown), although this may be obscured
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by the inherently long (Table 2) half-life of PAL in this strain. Importantly, PAL from R. graminis was quite stable during the bioreaction process. The PAL half-life and final PAL activity were superior to those observed for R. rubra at the 1- and 10-liter bioreactor scale. PAL activity in cells of R. graminis (wild type or mutant) harvested from bioreactors was generally 35 to 40% of the initial activity after approximately 90 h in a bioreactor. By comparison, R. rubra GX3243, R. glutinis IF00559 (19), and R. rubra SPA10 (3) cells generally retained 20% of their initial activity after 20 to 48 h under similar bioreaction conditions. In conclusion, R. graminis clearly possesses several desirable characteristics as a production strain. Mutants of R. graminis (e.g., GX6000) produce significantly higher PAL titers and specific activities than do other species of yeast (4, 6, 9, 15). L-Leucine acts synergistically with phenylalanine to further improve PAL formation. R. graminis fermentations do not require stringent temperature control and reduced oxygen tension as described by others to stabilize PAL (3, 15; M. A. Finkelman and H.-H. Yang, U.S. patent 4,584,273, April 1986). The most notable characteristic is the greater stability of R. graminis PAL during fermentation (four- to fivefold) and bioreaction (twofold) than R. rubra PAL. The inherent stability of R. graminis PAL may expand its potential applications to include medical uses such as the treatment of phenylketonuria disorders (10) and neoplastic tumors (5) and the rapid quantification of L-phenylalanine in blood (13). Currently, these applications are impractical, in part because of the instability of commercial PAL preparations. ACKNOWLEDGMENTS We thank Nils Adey for technical assistance, Jeff McGuire for critical review of the manuscript, and Lorraine Lorenz and Annette Seaborough for typing. LITERATURE CITED 1. Durham, D. R., C. G. McNamee, and D. B. Stewart. 1984. Dissimilation of aromatic compounds in Rhodotorula graminis: biochemical characterization of pleiotropically negative mutants. J. Bacteriol. 160:771-777. 2. Durham, D. R., and P. V. Phibbs. 1982. Fractionation and characterization of the phosphoenolpyruvate: fructose 1-phosphotransferase system from Pseudomonas aeruginosa. J. Bacteriol. 149:534-541. 3. Evans, C. T., D. Conrad, K. Hanna, W. Peterson, C. Choma, and M. Misawa. 1987. Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to L-phenylalanine. Appl. Microbiol. Biotechnol. 25:399405.
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4. Evans, C. T., K. Hanna, D. Conrad, W. Peterson, and M. Misawa. 1987. Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of L-phenylalanine. Appl. Microbiol. Biotechnol. 25:406-414. 5. Fritz, R. R., D. S. Hodgins, and C. W. Abell. 1976. Phenylalanine ammonia lyase. Induction and purification from yeast and clearance in mammals. J. Biol. Chem. 251:4646-4650. 6. Gilbert, H. J., and M. Tully. 1982. Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides. J. Bacteriol. 150:498-505. 7. Hamilton, B. K., H.-Y. Hsiao, W. E. Swann, D. M. Anderson, and J. J. Delente. 1985. Manufacture of L-amino acids with bioreactors. Trends Biotechnol. 3:64-68. 8. Hegeman, G. D., and R. T. Root. 1976. The effect of a nonmetabolizable analog on mandelate catabolism in Pseudomonas putida. Arch. Microbiol. 110:19-25. 9. Hodgins, D. S. 1971. Yeast phenylalanine ammonia-lyase. J. Biol. Chem. 246:2977-2985. 10. Hoskins, J. A., G. Jack, R. J. D. Peiris, D. J. T. Starr, H. E. Wade, E. C. Wright, and J. Stern. 1980. Enzymatic control of phenylalanine intake in phenylketonuria. Lancet i:392-394. 11. Kalghatgi, K. K., and P. V. Subba Rao. 1975. Microbial Lphenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani. Biochem. J. 149:65-72. 12. Kishore, G., M. Sugumaran, and C. S. Vaidyanathan. 1976. Metabolism of DL-(±)-phenylalanine by Aspergillus niger. J. Bacteriol. 128:182-191. 13. Koyama, H. 1984. A simple and rapid enzymatic determination of L-phenylalanine with a novel L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501. Clin. Chim. Acta 136:131-136. 14. Marusich, W. C., R. A. Jensen, and L. 0. Zamir. 1981. Induction of L-phenylalanine ammonia-lyase during utilization
of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis. J. Bacteriol. 146:1013-1019. 15. Nakamichi, K., K. Nabe, S. Yamada, and I. Chibata. 1983. Induction and stabilization of L-phenylalanine ammonia-lyase activity in Rhodotorula glutinis. Eur. J. Appl. Microbiol. Biotechnol. 18:158-162. 16. Onishi, N., K. Yokozeki, Y. Hirose, and K. Kubota. 1987. Enzymatic production of L-phenylalanine from trans-cinnamic
acid by Endomyces lindneri. Agric. Biol. Chem. 51:291-292. 17. Sato, T., F. Kiuchi, and U. Sankawa. 1982. Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds. Phytochemistry 21:845-850. 18. Wick, J. F., and J. E. Willis. 1982. Phenylalanine-dependent de novo synthesis of phenylalanine ammonia-lyase from Rhodotorula glutinis. Arch. Biochem. Biophys. 216:385-391. 19. Yamada, S., K. Nabe, N. Izuo, K. Nakamichi, and I. Chibata. 1981. Production of L-phenylalanine from trans-cinnamic acid with Rhodotorula glutinis containing L-phenylalanine ammonialyase activity. Appl. Environ. Microbiol. 42:773-778.
