Amino Acid Overproduction and Catabolic Pathway Regulation in

Blotechnol. Prog. 1994, IO, 372-376

372

Amino Acid Overproduction and Catabolic Pathway Regulation in Saccharomyces cerevisiae Enrique Martinez-Force**+ and Tahla Benitez Departamento de Genbtica, Facultad de Biologla, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain

To determine whether blocking a degradative pathway leads to amino acid accumulation, the internal free concentrations of threonine, methionine, and related amino acids have been measured in a strain of Saccharomyces cereuisiae and its mutants lacking either theL-threonine deaminase enzyme (EC4.2.1.16) coded for by theILVl gene (ilv-mutants), that coded for by the CHAl gene (cha- mutants), or both threonine deaminase enzymes (ilvcha- mutants). Whereas maximal accumulation of internal free amino acids occurred in the double mutants ilrcha-, i l v single mutants displayed amino acid concentrations higher than those of either the wild type or the cha- single mutant. On the other hand, when these enzymes were measured in threonine and methionine overproducer mutants of an industrial strain of Saccharomyces cereuisiae, results indicated, in most cases, lower activities than those of the wild type, although there was not a total correlation between maximal threonine and/or methionine accumulation and minimal enzymatic activities. Results point to the isolation of strains that accumulate essential amino acids by blocking their degradative pathway.

Introduction Yeast biomass is used to supplement human and animal diets, with baker’s yeast (Saccharomycescerevisiae) being regarded as a specializedform of single-cellprotein (Halasz and Lasztity, 1991). The production of a biomass with increased methionine, threonine, and tryptophan content constitutes a very effective means for supplementing food and feed deficient in these amino acids. Methionine is one of the most important essential amino acids, not only as an important building block of body proteins but also as an important methyl group donor (Jones and Fink, 1982; Niderberger, 1989). Also, the deficient methionine, threonine, and tryptophan supply contributes to the frequent incidence of various diseases, such as hepatic disease, anemia, etc. (Halasz and Lasztity, 1991). Proposals have been made to increase the protein content of bread by adding inactive yeast in order to improve the nutritional value of the lysine-,threonine-, and methionine-deficient cereal protein (Halasz and Lasztity, 1991; Niderberger, 1989). However, although the amino acid composition of different yeast strains varies with species and growing conditions (Halasz and Lasztity, 1991;Martinez-Forceand Benltez, 1992a,b, 1993), it is valid that the methionine, threonine, and tryptophan content is low for all yeasts (Jones and Fink, 1982;Martinez-Forceand Benltez, 1992a, 1993). Three potential methods can be used for the production of yeast biomass with an elevated content of specificamino acids: (1) Selection of mutants producing higher levels of methionine, threonine, or tryptophan. In this sense, mutants of Candida petrophilum and Candida tropicalis have been described, which possess a methionine content

* Address correspondenceto Enrique MarthesForce, Department of Biochemistry,419Roger Adams Laboratory, 600S. Mathews Ave., Urbana, IL 61801. Telephone: 217-244-2754.FAX 217-244-5858. E-mail: [email protected]. + Present address: Department of Biochemistry, 419 RAL, University of Illinois, Urbana, IL 61801.

about 40% higher than the wild type (Komatsu et al., 1974). This increase was entirely the result of the rise of free methionine concentration. Also, amino acid overproducer mutants of Saccharomyces cerevisiae have been selected in continuousculture that produce almost 40 times more threonine and 160 times more methionine than the wild type (Martlnez-Force and Benltez, 1992a, 1993). (2) Optimization of the conditions of fermentation. It has been demonstrated that environmental conditions, such as the medium composition,phase of growth (Praekelt and Meacock, 19921, growth rate (Kiss and Stephanopoulus, 1992;Martinez-Forceand Benltez, 1992b),respiratory or fermentative metabolism (Martfnez-Forceand Benftez, 1992b; Verduyin et al., 1992; Zitomer and Lowry, 1992) temperature, etc., considerably influence the free amino acid pool of yeast strains, so that this concentration can increase up to 12 times depending on the amino acid by, for instance, varying the growth rate in continuous culture (Martlnez-Force and Benltez, 1992b). (3) Genetic engineering of yeasts. This method is applicable only when there is vast knowledge of the key enzymes involved in regulation of the amino acid biosynthetic pathway (Niderberger, 1989; Ramos et al., 1991). Frequently, the key enzyme of a specific amino acid synthesis is derepressed in feedback-resistant mutants (Halasz and Lasztity, 1991; Martlnez-Force and Benltez, 1993;Ramos and Calderh, 1992). The enzyme is resistant against, for instance, threonine or methionine and their toxic analogues, hydroxynorvaline or ethionine (Ramos and Calderh, 1992). Hence, mutants resistant to ethionine and hydroxynorvaline are able to produce methionine and threonine in excess (Martlnez-Force and Benltez, 1992a; Ramos and Calderh, 1992). Accumulation of free amino acid can also be obtained by blocking the catabolic pathway, so that the amino acid is not, or is very slowly, degraded as occurs with methionine accumulation, which has been found in the case of a low S-adenosylmethioninesynthetase level (Halasz and Lasztity, 1991; Martlnez-Force and Benftez, 1993). In these cases, the work associated with the increase in amino acid

8756-7938/94/30 10-0372$04.50/0 0 1994 American Chemical Society and American Institute of Chemical Engineers

Bbtechtwl. hog.., 1994, Vol. 10, No. 4

373

Aspart ate

4

Homose rine

-(....)-

i

Methionin e &A: SH

Threonine>-

7hreoni n e &amin a se

Propionil- CoA

a-keto-butyrate

Serine hydroxymeth ylpan sferase

NAD+

NADH

Glycine 2-amino- 3- keto butyrate

Ac etaldeh y &

Aldehyde dehydrogenase

I

Ac etil- CoA

Serine Serine deaminase Pyruvate

Deczr b y l a r i m

Amino-aceton e

I

Deaminaticn

t

Meth yl-g lyoxal

Dlaitate

Figure 1. Pathways of threonine catabolism and ita relationship with other amino acids. content should preferably start with the selection of existing strains or their mutants on the basis of their high amino acid contents. In yeasts, threonine catabolism is carried out by either of three enzymatic activities (Figure 1):threonine deaminase (EC 4.2.1.16), which converts threonine into a-ketobutyrate, threonine dehydrogenase(EC 1.1.1.103),which converts threonine into 2-amino-3-ketobutyrate, and the serine hydroxymethyl transferase (EC 2.1.2.1), which transforms threonine into glycine (Jones and Fink, 1982). Furthermore, threonine deamination or the dehydration reaction is carried out by either of two different threonine deaminase enzymes: the anabolic one coded for by the ILVl gene, which is the first enzyme in the biosynthesis pathway of isoleucine, and a second one, which is catabolic, is coded for by the CHAl gene and allows yeast cells to grow with serine or threonine as the only nitrogen source (Petersen et al., 1983; Ramos and Wiame, 1982). In order to select an amino acid overproducer yeast of mostly methionine and threonine, the internalamino acid pool and the enzymes of the catabolism of threonine have been determined in this work in an industrial strain of Saccharomyces cerevisiae, in its amino acid overproducer mutants, and in mutants lacking either ILVI, CHA1, or both gene products. The possible correlation between catabolic enzyme deregulation and amino acid accumulation is discussed. Materials and Methods Strains. Isogenic Saccharomyces cerevisiae strains 8655C (MATa chal), 8723c (MATa ilvl), and 8736b (MATa chal ilvI) were generously provided by Dr. F. Ramos (Microbiologie, Faculte des Sciences, Universite Libre de Bruxelles, Brussels, Belgium). The mutation ilvl

was previously obtained by Dr. Marcelle Grenson. The strain REMF5 (MATu) was a spontaneous revertant obtained from strain 8723~. saccharomyces cerevisiae IFI256, a highly fermentative industrial strain, was a gift of Dr. V. Arroyo, (Instituto de FermentacionesIndustriales, Madrid, Spain). The threonine and methionine overproducer mutants ETHCC1-ETHCC8, HNVBC4, and HNVBC5 were isolated in continuous culture from strain IF1256 as described elsewhere (Martinez-Force and Benltez, 1992a). Enzymes and Chemicals. Acetonitrile was obtained from Fluka, A.G. (Buchs, Switzerland); methanol was purchased from Probus (Barcelona, Spain); tetrahydrofuran, a-ketobutyrate, pyridoxal phosphate, and 2,4dinitrophenyIhydrazine were from Merck, A.G. (Darmstadt, Germany);sodium phosphate and sodium borate were from Panreac (Barcelona,Spain); D,L-homoserinewas from Janssen Chimica (Belgium); lactate dehydrogenase (EC 1.1.1.27) (LDH), pyruvate kinase (EC 1.1.1.27) (PK) from rabbit muscle, phosphoenolpyruvate (tricyclohexylammonium salt), o-phthalaldehyde, j3-mercaptoethanol, individual crystalline amino acid standards or mixtures thereof, 4-(2-hy&oxyethyl)-l-piperazineethanesulfonic acid (sodium salt) (Hepes), adenosine 5’-triphosphoric acid (sodium salt), dithiothreitol (DTE), ethylenediaminetetraacetate (disodium salt) (EDTA), nicotinamide adenine dinucleotide (oxidized,NAD+,and reduced, NADH, forms) (sodium salt), protamine sulfate, as well as all other chemicals used were purchased from Sigma Chemical Co. (St. Louis, MO). Media. Yeasts were grown in either beet molasses (72 % sucrose) obtained from Uni6n Alcoholera Espaiiola, S.A. (Granada, Spain), diluted 20 times (3.6% sucrose, M medium), or in Y P medium (0.5%Difco yeast extract and

Bbtechnol. Rog., 1994, Vol. 10, No. 4

374

1% bactopeptone) supplemented with either 2 % glucose

(YPD), 2% fructose (YPF), or 3% glycerol (YPG). Occasionally,biotin (0.5mg/L, MI3medium), diammonium phosphate (0.5 g/L, MA medium), or both (0.5mg/L biotin and 0.5 g/L diammonium phosphate, MAB medium) were added to the molasses. A minimal medium (0.17% Difco yeast nitrogen base without amino acids and with 0.5% ammonium sulfate), prepared with either 2 % glucose (SD) or 3% glycerol (SG), was also used. When necessary, isoleucine at a final concentration of 30 mg/L was added to the minimal medium. CultureConditions. The strains were routinely grown at 30 "C in 10-mL tubes containing 3 mL of SD or this medium supplemented with isoleucine until they reached stationary phase. At this point, 0.5 mL was inoculated into 50-mL Erlenmeyer flasks containing 25 mL of either SD or SD with the appropriate amino acid concentration, and the cultures were incubated at 30 "C in a shaker. After inoculation, samples were taken periodically, and the absorbance at 660 nm ( A m ) was determined until the culture reached the stationary phase. An exponential increase in A m between 0.1 and 0.5 was used to determine the growth rate, p. Previously, a linear relationship between cell number and A m , ranging from 0.1 to 0.5, was established. Cell-FreeExtract Preparation. Cell-free extracts and partial purifications of the enzymes were prepared according to Ramos et al. (Ramos et al., 1991). Assay of Threonine Dehydrogenase. Threonine dehydrogenase activity (EC 1.1.1.103) was assayed routinely by monitoring NADH production (as the increase in OD at 340 nm) (Burr et al., 19761, after mixing NAD+ in Hepes buffer with either cell-freeextract or semipurified extract and initiating the reaction by adding L-threonine to a final concentration of 40 mM. One unit of activity was defined as the amount of enzyme required to convert 1 pmol of NAD+ to NADH per minute at 30 "C. Assay of Threonine Deaminase. Threonine deaminase activity (EC 4.2.1.16) was measured by monitoring NADH consumption (as the decrease in OD at 340 nm) with the pyruvate kinase (PK)/lactate dehydrogenase (LDH) coupled assay (Burr et al., 1976;Theze et al., 1974), after mixing in Hepes buffer NADH and PK/LDH with either cell-free extract or semipurified extract and initiating the reaction by adding L-threonine to a final concentration of 60 mM. One unit of activity was defined as the amount of enzyme required to convert 1pmol of NADH to NAD+ per minute at 30 "C. Pyruvate Determination. Pyruvate was quantified to determine the threonine deaminase activity specifically coded for by the CHAl gene, which catabolizes the formation of one molecule of pyruvate from one molecule to serine. The same assay was used to determine a-ketobutyrate formation from threonine, catalyzed by either the anabolic threonine deaminase or the catabolic threonine (serine) deaminase (Ramos and Wiame, 1982). The activity was assayed by mixing pyridoxal phosphate and potassium phosphate buffer with EDTA and cell-free extract and initiating the reaction by adding serine to a final concentration of 40 mM. After incubation, the reaction was stopped with TCA, centrifuged, and newly incubated with 2,4-dinitrophenylhydrazine.Hydrazones were finally estimated at 520 nm after adding NaOH. The micromoles of pyruvate formed per milliliter and per minute were obtained after multiplying the OD (measured) by 0.74 (Ramos and Wiame, 1982). Internal Pool of Amino Acids. Primary amino acids were derivatized with o-phthalaldehyde (OPA) and /%mercaptoethanol and detected by absorbance at 340 nm. The derivatization mixture was prepared by mixing 4.5 mL of

Table 1. Internal Concentration of Threonine and Related Amino Acids (mM)and Specific Activity (units/g) of the Anabolic Threonine Deaminase (TD) and the Catabolic Threonine (Serine) Deaminase (STD) of the Wild-Type Strain REMF5 and the Isogenic Mutants 8655c (cha-), 8723c (ilv-1, and 8736b (cha-ilv) Cultivated in YPD at 30 O C . amino acid (mM)

REMF5

ASP Met Thr Ile Val Leu Ser G~Y Ala activity TD STD TDA(TD+STD)

2.3 1.1 6.0 4.2 3.7 3.4 7.2 16.1 20.8 1.35 0.42 1.95

strain 8655c 8723c (cha-) (ilv-)

8736b (chailv)

2.25 1.20 5.8 3.7 3.4 3.1 6.4 18.4 21.5

4.04 1.53 11.0 8.4 11.6 2.9 22.1 15.2 42.8

7.93 3.12 15.7 12.5 19.1 3.2 55.3 17.3 43.2

1.6 nd 1.6

ndb 0.34 0.34

nd nd nd

Results are the average of four experiments, with a standard deviation of 1.84-2.10%. nd, not detected.

sodium borate (0.4 M, pH 10) with 0.5 mL of o-phthalaldehyde (54 mg of OPA/mL of methanol), maintained at -20 "C and protected from the light, and 0.2 mL of 0-mercaptoethanol. The mixture was filtered through a 0.45-pm pore size Millipore filter and maintained at -20 "C. Amino acid samples (the filtered supernatant from boiled cell suspensions) were derivatized by mixing 25 pL of each with 75 pL of the derivatization mixture. After a 2-min incubation at room temperature, the derivatized samples were separated using an acetonitrile/phosphate buffer gradient (Martfnez-Force and Benftez, 1991) by reverse-phase high-performance liquid chromatography (HPLC), following methods described previously (Martlnez-Force and Benitez, 1991). Protein Determination. Protein concentration was determined according to the Bradford procedure (Bradford, 1976).

Results Amino Acid Pool in the i l v , cha-, and ilvchaMutants. As indicated before, in Saccharomyces cereuisiae, threonine can be metabolized by the threonine dehydrogenase, the serine hydroxymethyl transferase, and either the L-threonine deaminase coded for by the ILVl gene or the L-threonine (L-serine)deaminase coded for by the CHAl gene. Whereas the ILVl gene product is necessary to synthesize isoleucine,the CHAl gene product is induced only by high concentrations of either serine or threonine (Ramos and Wiame, 1982). When the internal concentrations of threonine, methionine, and related amino acids were measured in the control strain REMF5 and in the mutants 8723c (ilv-1, 8655c (cha-), and 873613 (ilv-cha-), maximal methionine and threonine concentrations (3.12 and 16 mM, respectively) were detected in the double mutant 8736b (Table 1). The ilv- mutant 8723c showed methionine and threonine concentrations of 1.53 and 11mM, respectively, whereas the control had 1.1 and 6 mM concentrations, respectively. The cha- mutant 8655c had concentrations similar to those of the control: 1.2 and 5.8 mM, respectively. Serine and threonine concentrations in the chamutants were also similar to those of the control; however, in the ilv- and the chailv- mutants these concentrations were substantially higher: 11and 15.7 mM threonine and 22.1 and 55.3 mM serine, respectively. This increase in threonine and serine concentrations would explain the

Biotechnol. Rog...1994, Vol. 10, No. 4

Table 2. Growth Rate (h-1) of the Industrial Strain of Saccbaromyces cerevisiae IF1256 and the Laboratory Isogenic Strains REMF5,8655c (cha-), 8723c (ilv-), and 87368 (cha-ilv-) Grown in Complete Medium (YPD), Minimal Medium Supplemented with Isoleucine (SD+Ile), Molasses (M), and Molasses Supplemented with Either Ammonium Phosphate (MA) or Biotin (MB). 8655c 8723c 8736b medium IF1256 REMF5 (cha-) (ilv-) (chailv-) 0.20 0.24 0.21 YPD 0.39 0.27 0.22 0.20 0.19 0.16 SD+Ile 0.31 0.03 0.02 0.02 M 0.11 0.03 0.05 0.04 0.04 0.05 MA 0.14 MB 0.11 0.03 0.03 0.03 0.03 4 Results are the average of three experiments, with a standard deviation varying between 0 and 0.02.

differences observed between the ilv- and the ilv-chamutants, since in the former the CHAl gene product was induced. The pools of Asp, Ile, Val, Ser, and Ala also showed great increases, especially in the ilvcha- mutant. Growth of the Different Strains in Laboratory and Semiindustrial Media. As Table 1shows, the internal concentration of essential amino acids such as methionine or threonine increases in ilv- and above in all cha-ilvmutants, but these mutants are unable to synthesize isoleucine when growing in minimal medium. However, in molasses, where industrial strains are usually grown, the mutants have a fairly high concentration of free amino acids (between 10and 400 pg/mL, depending on the amino acid) (Martinez-Force and Benitez, 19931,which could be directly used by the yeast strains. Growth rate, therefore, was measured in the strains grown in complete medium (YPD), in minimal medium supplemented with isoleucine (SD+Ile), and in semiindustrial medium (molasses)with or without an ammonium phosphate or biotin supplement. Maximal growth rate was shown in all casesby the industrial strain IF1256 (Table 2). This strain, however, as well as the amino acid overproducing mutants, which grow on molasses at growth rates similar to that of their parental IF1256 strain (data not shown), reduced its growth rate almost 4 times when it was grown in molasses, as compared to YPD. Similarly, the laboratory strain REMF5 and the mutants 8655c, 8723c, and 8736b also considerably reduced their growth rates in molasses, with little difference between the prototrophic strain REMF5 and the other strains. Results therefore point to the isolation of cha-ily mutants from industrial strains, which will possibly behave as the parental strain in industrial media, increasingtheir internal amino acid concentrations. Threonine Degradation in the Industrial Strain IF1256 and Its Amino Acid Overproducer Mutants. Amino acid overproducer mutants of the strain IF1256 were isolated in continuous culture by increasing the concentrations of toxic amino acid analogues such as ethionine and hydroxynorvaline (Martinez-Force and Benftez, 1992a). The amino acid overproduction could be the result of the alteration of both the biosynthetic pathway enzymes, such as aspartate kinase and homoserine kinase (Martfnez-Force and Benltez, 1993),losing the regulation by feedback inhibition, and the inactivation of the degradation pathway enzymes, giving rise to amino acid accumulation. When threonine deaminase was measured in the strain IF1256 and the mutants (Table 3), the activity was, in most cases (withtwo exceptions: ETHCCl withan specific activity similar to and ETHCC2 with an specific activity slightly higher than that of the wild type), lower than that of the wild type. Results therefore indicated that, in addition to the biosynthetic pathway alteration, threonine

376

+

Table 3. Threonine Deaminase (TDA = TD STD) and Threonine Dehydrogenase (TDH) Activities (units/g of protein) of the Saccbaromyces ceredsiae Strain IF1256 and Its Threonine and/or Methionine Overproducing Mutants and Intracellular Concentration of Threonine and Methionine in the Mutants as Compared with the Wild Type, Whose Concentrations Are Considered as the Unit strain IF1256 ETHCCl ETHCC2 ETHCC3 ETHCC4 ETHCC5 ETHCC6 ETHCC7 ETHCC8 HNVBC4 HNVBC5

activities TDA TDH 2.31 0.26 2.34 0.24 3.25 0.20 0.48 0.23 1.05 0.24 0.90 0.25 1.41 0.20 1.20 0.25 0.82 0.23 0.90 0.20 0.40 0.19

internal amino acid concentration Thr Met 14 10 1.8 11.5 2.9 16.0 2.9 25.5 2.9 32.5 2.7 43.5 2.0 51.0 2.6 163.0 13.7 15.5 4.0 30.0 37.0 17.5

4 The absolute values of the wild type are (mM) Thr (0.6)and Met (0.2). Results are the average of four experiments, with standard deviations between 0.004 and 0.006.

accumulation in the mutants was due to the decrease in the enzymatic activity of the degradative enzyme, threonine deaminase. However, as Table 3 also shows, there was not a close correlation between threonine and methionine accumulation and the decrease in threonine deaminase activity. With regard to the threonine dehydrogenase enzyme, the specific activity was very similar in the wild type and in the mutants, indicating that this enzyme probably does not play a significant role in threonine and methionine overproduction. Discussion This work has studied whether blocking an amino acid degradative pathway leads to the accumulation of the amino acid. For instance, the threonine deaminase enzyme coded for by the IL VI gene seems to play a very significant role in the degradation of threonine and is, at the same time, the first enzyme involved in the isoleucine biosynthetic pathway (Petersen et al., 1983; Ramos and Wiame, 1982). Mutants lackingthis activity ( i l r mutants) become auxotrophs of isoleucine, but in the presence of high concentrations of threonine and/or serine, the threonine deaminase coded for by the CHAl gene is induced and the ilv- mutants are able to grow with threonine and/or serine as the only nitrogen source (Ramos and Wiame, 1982). In the presence of a preferred nitrogen source such as ammonia, the biosynthesis of enzymes required for the catabolism of other nitrogen compounds is usually repressed (Praekelt and Meacock, 1992). However, with regard to the threonine deaminase, it has been described that the addition of polyamine, an activator of AMP deaminase, resulted in the increase in ammonia concentration that stimulates the activity of the yeast threonine deaminase enzyme (Yoshino and Murakami, 1981) and the synthesis of isoleucine and valine. Furthermore, a t least the threonine deaminase coded for by the ILVl gene is derepressed under conditions of amino acid starvation, suggesting that the ILVl gene is under the general control of amino acid biosynthesis (Debourg and Pierod, 1990) and is transcriptionally regulated, since by RNA hybridization (Northern analysis) a 5-10-fold decrease in the threonine deaminase mRNA level was observed when minimal medium with leucine was supplemented with isoleucine and valine (Petersen et al., 1983). Whereas the anabolic enzyme coded for by the IL V1gene is an allosteric enzyme sensitive to feedback inhibition by isoleucine, no

Blotechnol. Rag., 1994, Vol. 10, No. 4

976

control of the activity of the catabolic enzyme coded for by the CHAl gene, which behaves as a typical Michaelian enzyme, could be demonstrated (Ramosand Wiame, 1982). In this study, preliminary data indicated that the threonine deaminase coded for by the CHAl gene could not be induced in the presence of ammonia salt and low concentrations of threonine and serine (data not shown), but could be induced in complete medium, both in the control and the ilv- mutants (Table 11,and in media with ammonia salt as a nitrogen source in the threonine overproducer mutants (Table 3). The results therefore indicated the CHAl gene can be induced by high internal concentrations of threonine and serine, regardless of the presence of ammonia. In Table 1,the highest concentration of threonine and methionine was observed in the double mutant ilv-cha-. The high concentration of serine detected was probably the reason for the induction of the CHAl gene in the ilvmutant and, therefore, the reason for the difference in internal amino acid concentrations between the i l v and ilv-cha- mutants. Together with the increase in methionine and threonine concentrations observed in the ilv-cha- mutant, other amino acids such as Asp, Ile, Val, Ser, and Ala also showed considerable increases. Whereas the increase in serine concentration could be attributed to the lack of the functional product of the CHAl gene (Ramosand Wiame, 1982), threonine accumulation in the ilv-cha- mutant inhibits its own biosynthetic pathway (Martfnez-Force and BenItez, 1993),thus giving rise to aspartate accumulation. This, in turn, produces pyruvate accumulationviathe TCA pathway (Jones and Fink, 1982). The increase in pyruvate concentration results in an increase in all of those pyruvatederived amino acids, such as valine, alanine, and isoleucine (Jones and Fink, 1982). Fermentations that aim for high biomass production, for example, the production of amino acid-enriched yeast biomass, are generally based on cheap, complex substrates such as molasses that contain variable amounts of organic nitrogen growth factors and mineral salts (Martfnez-Force andBenItez,1993;Praekelt and Meacock, 1992). The fairly high concentration of free amino acids found in molasses (Martlnez-Force and Benltez, 1993) makes feasible the possibility of using auxotrophic ilv-cha- mutants able to accumulate threonine and methionine for industrial purposes. However, when the strains were cultivated in several media (Table 21, they reduced their growth rate between 3 and 8times in molasses,as compared to complete YPD medium. The best results were obtained with strain IF1256 (Table 2) and the amino acid overproducing mutants (data not shown), pointing to the possibility of isolating ilv-cha- mutants from industrial strains for applied purposes. Although most threonine and methionine overproducer mutants (Table 3) had threonine deaminase activities lower than that of the wild type, there was not a close relationship between amino acid overproduction and enzyme activity inactivation, indicating that amino acid accumulation can be reached partly by blocking amino acid pathway degradation, although higher increases can be obtained by deregulatingthe biosynthetic pathway, as has previously been observed (Martinez-Force and Benltez, 1992a, 1993; Ramos and Calderbn, 1992).

Acknowledgment This work was supported by CAICYT Project No. BI090-0504.

Literature Cited Bradford,M. M. Arapid and sensitivemethod for thequantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. Burr, B.; Walker, J.; Truffe-Bachi,P.; Cohen, G. N. Homoserine kinase from Escherichia coli K12. Eur. J.Biochem. 1976,62, 519-526. Debourg, A.; Pierod, A. The regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae: influence of the ZLVl gene doses and of leu3 and gcn4 regulatory mutations. Yeast (15th Znt. Conf. Yeast Genet. Mol. Biol.) 1990,Special Issue, S263. Halasz, A.; Lasztity, R. Use of yeast biomass in food production. CRC Press Inc.: Boston, 1991. Jones, E. W.; Fink, G. R. Regulation of amino acidsandnucleotide biosynthesis in yeast. In The molecular biology of the yeast Saccharomyces: Metabolismand gene expression;Strathem, J. H., Jones, E. W., Broach, J. R., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1982; pp 181230. Kiss,R. D.; Stephanopoulos,G. Cultureinstability of auxotrophic amino acid producers. Biotechnol. Bioeng. 1992,40, 75-85. Komatsu, K.; Yamada, T.; Kdaira, R. Isolation and characteristics of pool methionine-rich mutants of Candida sp. J. Fermen_t.Technol. 1974,52,93-98. Martfnez-Force, E.; Benftez, T. Separation of o-phthaladehyde derivatives of amino acids of the internal pool of yeast by reverse-phaseliquid chromatography. Biotechnol. Tech. 1991, 5, 209-214. Martinez-Force, E.; Benftez, T. Selection of amino-acid overproducer yeast mutants. Curr. Genet. 1992a, 21,191-196. Martfnez-Force,E.; Benftez,T. Changesin yeast amino acid pool with respiratoryversusfermentativemetabolism. Biotechnol. Bioeng. 1992b, 40,643-649. Marthez-Force, E.; Benftez,T. Regulation of aspartate-derived amino acid biosynthesisin the yeast Saccharomycescerevisiae. Cum. Microbiol. 1993,26, 313-322. Niderberger,P. Amino acids production in microbialeukaryotes and prokaryotes other than coryneforms. In Microbial products: new approaches;Baumberg,S.,Hunter, I., Rhodes, M., Eds.; CambridgeUniversityPress: Cambridge,U.K., 1989; pp 1-24. Petersen, J. G. L.; Holmberg, S.; Nilsson-Tillgren, T.; KiellandBrandt, M. C. Molecular cloning and characterization of the threonine deaminase (ZLVl)gene of Saccharomycescerevisiae. Carlsberg Res. Commun. 1983,48,149-159. Praekelt, U. M.; Meacock, P. A. MOLl, a Saccharomyces cereuisiae gene that is highly expressed in early stationary phase during growth on molasses. Yeast 1992,8, 699-710. Ramoe, F.; Wiame, J. M. Occurrence of a catabolic L-serine (Lthreonine) deaminase in Saccharomyces cerevisiae. Eur. J. Biochem. 1982,123,571-576. Ramos,C.; Delgado,M. A.; Calderdn, I. L. Inhibition by different aminoacids of the aspartate kinase and the homoeerinekinase of the yeast Saccharomycescerevisiae. FEBS Lett. 1991,278, 123-1 26. Ramos, C.; Calderdn, I. L. Overproduction of threonine by Saccharomyces cerevisiae mutants resistant to hydroxynorvaline. Appl. Environ. Microbiol. 1992,58, 1677-1682. Them,J.; Kleidman,L.;Saint-Girons, I. Homoserinekinase from Escherichia coli K12 properties, inhibition by L-threonine and regulation biosynthesis. J.Bacteriol. 1974,118,577-581. Verduyin, C.; Poetma, E.; Scheffers, W. A.; van Dijken, J. P. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culturestudy on the regulation of respiration and alcoholic fermentation. Yeast 1992,8,501-517. Yoshino, M.; Murakami, K. The effect of spermine on the interaction of AMP deaminase with threonine dehydratase activity in yeast. Biochim. Biophys. Acta 1981,672,313-316. Zitomer, R. S.; Lowry, C. V. Regulation of gene expression by oxygen in Saccharomyces cerevisiae. Microb. Rev. 1992,56, 1-11.

Accepted March 7, 1994.@ @

Abstract published in Advance ACS Abstracts, April 15,1994.