Agric. Biol Chem., 51 (1), 129-138, 1987 129

Pyruvate Formation and Sugar in an Amino Acid-Producing Bacterium, Brevibacterium flavum Michiko Mori and Isamu Shiio Central Research laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki, Kanagawa210, Japan Received July 2, 1986

A Brevibacteriumflavum mutant lacking pyruvate , No. 70, grew on , fructose and sucrose as well as the original wild strain did, but wasunable to grow on ribose or gluconate unless pyruvate was added. Mutants that required pyruvate for growth on ribose were derived directly from the wild strain. Manyof them were completely or partially defective in pyruvate kinase activity. These pyruvate kinase mutants were also unable to grow on gluconate. Aphosphoenolpy- ruvate (PEP) : sugar system (PTS) was found in B. flavum, which catalyzed the formation ofpyruvate and sugar from PEPand sugar. The system required Mg2+ , acted on glucose, fructose, mannose, glucosamine and 2-deoxyglucose, and existed in the cells grown on any of the carbon sources tested. Cells grown on fructose, mannitol and sucrose, however, exhibited higher PTSactivities on fructose than those grown on others. Glucose PTSactivity was about 20- fold stronger than that of . Other sugar metabolic , inducible mannitol dehydrogenase, , and maltase, as well as constitutive invertase were also detected. Oxaloacetate decarboxylase and malic , which also catalyzed the pyruvate formation, were found in B. flavum, but the latter activity was very low in cells grown on glucose. The levels of these enzymesin pyruvate kinase mutants unable to growon ribose or gluconate derived from the wild strain were almost identical to those in the wild-type strain.

Since phosphoenolpyruvate (PEP) is a suggesting that enzymesother than PKmight branch-point intermediate in the sugar me- serve to form sufficient pyruvate for growth. tabolism for amino acid biosynthesis, studies The present study revealed that the on enzymes catalyzing PEP metabolism seem PEP : sugar phosphotransferase system (PTS) interesting, especially for an amino acid- as well as PK significantly contribute to the producing bacterium, Brevibacterium flavum. pyruvate formation during growth on glucose, Studies on PEP carboxylase1} which catalyzes whereas only PK is responsible for the for- the formation of oxaloacetate, an intermediate mation during growth on sugars which are not in amino acid biosynthesis, and on pyruvate metabolized via the PTS reaction. kinase (PK)2) which catalyzes the first step of degradation of PEP to CO2 were reported MATERIALS AND METHODS previously. Pyruvate produced from PEP via the PK Chemicals. PEP, ADPand ATP were purchased from reaction is not only oxidized to yield CO2but Sigma Chemical Co., dithiothreitol from Calbiochem, and NAD, NADHand NADPfrom Boehringer Mannheim also used for the biosynthesis of various essen- GmbH.All the enzymes used for enzyme assays and for tial cell constituents. The growth on glucose of the metabolite determinations were obtained from the mutants lacking PK, however, was substan- latter company. tially identical to that of the wild-type strain, Abbreviations: PK, pyruvate kinase; PTS, phosphotransferase system; LDH,; PEP, phosphoenolpyruvate; FBP, fructose 1, 6-bisphosphate; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; CTAB, cetyltrimethylammonium bromide. 130 M. Mori and I. Shiio

Bacterial strains and culture media. Brevibacterium fla- assay, except that the cells for oxaloacetate decarboxylase vum No. 2247 (ATCC14067) (wild strain) and an and malic enzyme were cultured in a glucose medium, aspartate-producing mutant lacking PK, No. 70,3) were Medium34, supplemented with 300jug/liter of biotin for used. Medium41, a modification of Medium30,4) was 24hr, and in Medium 29 containing 40g/liter of glucose composed of 20g of glucose, 10g of ammoniumsulfate, and 15 g/liter of ammoniumsulfate instead of glutamate 3g ofurea, 1g ofKH2PO4, 0.4gofMgSO4à"7H2O, 0.5g of for 40 hr, respectively, and that the buffer for oxaloacetate NaCl, lOmg of FeSO4-7H2O, 8mg of MnSO4-4H2O, decarboxylase was supplemented with 0.5 mammonium 50f.ig ofd-biotin, 200/*g ofthiamineà"HC1 and 20g ofagar sulfate. in a total volume of 1 liter, adjusted to pH 7.0 with KOH, and sterilized at 115°C for lOmin. The composition of Enzyme assay Medium 135) was the same as that of Medium 41 except i) Sugar . Glucokinase was assayed by measur- that NaCl and agar were not added and that the con- ing glucose 6-phosphate (G6P) formation according to the centrations of J-biotin and thiamine-HC1 were 30 and method of Anderson and Kamel,9) while all other sugar 100/zg per liter, respectively. NaOHwas used for pH kinases were assayed by measuring ADPformation as adjustment. The complexmediacontaining polypeptone, follows. The reaction mixture contained 50mMTris-HCl yeast extract and glucose, Medium 75) and Medium 27,6) buffer, pH 7.5, 10mM sugar, 3.3mM ATP, 5mMMgCl2, and the synthetic media, Medium296) and Medium34,3) 2mMPEP, 0.15mM NADH, 10/ig ofPK, 10^g oflactate were reported in the previous papers. dehydrogenase (LDH) and enzymein a total volume of 1.5ml. The increase in absorbancy at 340nm was mea- Enzyme preparation sured. Control experiments were performed without sugar i) Sugar kinases and mannitol dehydrogenase. Crude substrates. Gluconokinase was also assayed by measuring enzyme extracts were prepared from cells cultured for 6-phosphogluconate formation with the same reaction 16 hr in Medium 13 containing the indicated sugar instead mixture as above, except that 0.4mMNADPand 5 /.ig of 6- of glucose, except that the culture times for the ribose and phosphogluconate dehydrogenase were added instead of mannitol media were 24 and 40 hr, respectively. Cells were PEP, NADH, PK and LDH. harvested by centrifugation, washed twice with 0.2% KC1, ii) Mannitol dehydrogenase. This enzyme was assayed suspended in 0.1m Tris-HCl buffer, pH 7.5, and then by measuring the increase in absorbancy at 340nm of the subjected to sonic disruption at 10Kc for 20min. After reaction mixture, which contained 50mMsodium bicar- centrifugation of the sonicate at 170,000 x g for 30 min, the bonate buffer, pH 10.0, 10mMmannitol, 0.5mM NADand supernatant was gel filtered through a Sephadex G-25 enzyme. column with the same buffer. iii) Sugar . Sucrose and ii) Sugar andphosphorylases. Crude enzyme maltose phosphorylase were assayed by the method of extracts were prepared as described above, except that Mieyal,10) using sucrose and maltose, respectively, as 0.1m potassium phosphate buffer, pH 7.0, was used substrates. instead of Tris-HCl buffer. iv) Sugar hydrolases. Invertase and maltase were de- iii) PTS. Toluenized cells were used for the PTS termined by measuring glucose formation according to the assay.7) Cells were cultured, harvested, washed as de- method of Goldstein and LampenU)for invertase. scribed above and then resuspended in a 0.1 mTris-HCl v) PTS. This activity was assayed by measuring sugar- buffer, pH 7.5, supplemented with 10% polye.thylene- dependent pyruvate formation by the following procedure. glycol and 30 him dithiothreitol. The cells were decryptified The reaction mixture contained 100 mMTris-HCl buffer, by adding 0.01ml of toluene to lml of the suspension, pH 7.5, 10mM PEP, 5mM MgCl2, 30mM dithiothreitol, followed by vigorous agitation for 1 min with a mixer, 10mM sugar and 20^1 of a toluenized cell suspension Taiyo S-5F. The toluenized cell suspension was washed whose absorbancy at 562nm was 0.5 after 250-fold di- with and then resuspended in the same buffer. lution. The mixture without sugar (total volume, 0.45 ml) iv) PK. Crude enzyme extracts for the PK assay were was preincubated at 30°C for lOmin. The reaction was prepared by sonication as described previously3) from cells started by the addition of 0.05ml of 100mM sugar and cultured for 16hr in Medium 27. Cetyltrimethyl- stopped by adding 0.1 ml of 30% metaphosphoric acid ammoniumbromide (CTAB)-treated cells8) were also after lOmin incubation at 30°C. After centrifugation, used for the PK assay. Washed cells prepared by the pyruvate produced in the supernatant was assayed. The above procedure and with an absorbancy at 562nm of composition of the assay mixture was 0.5 m TES-NaOH 0.7 after 26-fold dilution were treated with 0.6 mMCTAB buffer, pH 7.5, 0.15mM NADH, 50jug ofLDH and 0.1 ml in 0.1 m potassium phosphate buffer, pH 7.5, at 37°C for of the supernatant of the PTS reaction mixture in a final 10 min, and then resuspended in 0. 1 m Tris-HCl buffer, pH volume of 2.1 ml. Before and after addition of LDH,the 7.5, after centrifugation. absorbancy at 340nmwas measured. v) Oxaloacetate decarboxylase and malic enzyme. vi) PK. Two assay methods (A and B) were used to Crude enzymeextracts for the assay of these enzymeswere assay PK. Method A involving crude enzyme extracts was prepared by the same procedure as for the sugar kinase described previously.3) The composition of the reaction Pyruvate Formation and Sugar Metabolism in B. flavum 131 mixture for Method B with CTAB-treated cells was the PTS. Two distinct PKs, a constitutive AMP- same as that for Method A but without NADHand LDH. activated enzyme and an inducible FBP- After the reaction had been carried out at 30°C for 10 min, dependent one, have been identified in the mixture was placed in an ice bath and the cells were removed by centrifugation. Pyruvate formed was de- .14) In a partially purified termined as described above. preparation of B. flavum, PKwas found to vii) Oxaloacetate decarboxylase. The reaction mixture be activated by AMP but not by FBP.2) for the oxaloacetate decarboxylase assay contained 50 rriM Furthermore, inducible FBP-dependent PK TES-NaOH buffer, pH 8.0, 10mMMnSO4, 10mMoxa- was undetectable in the crude enzymeextract loacetate and enzymein a final volume of 1.0ml. The reaction was started by the addition of oxaloacetate after of cells grown on glucose, as shown in Table I. 5 min preincubation at 30°C, allowed to proceed for 5 min On the other hand, oxaloacetate decarboxylase at 30°C, stopped by the addition of 0.2ml of 30%meta- activity was considerably high in the wild-type phosphoric acid, and then cooled in an ice bath. Pyruvate strain. A PK mutant, No. 70, showed only produced in the reaction mixture was measuredas de- 10% of the parental activity. Malic enzyme scribed above. viii) Malic enzyme. This enzyme was assayed by activated by K+ was present at a high level in measuring the absorbancy at 340nm of the reaction the wild strain grown on glutamate (the data mixture, which contained 33 mMTris-HCl buffer, pH 7.5, will be presented in another report), but was 33mM malate, 0.27mM NADP, 1 mMMnCl2, 200mM KC1 essentially absent in the mutant. Since the and enzyme in a final volume of 1.5ml. enzymeactivity after growth on glucose was Isolation ofpyruvate kinase-deficient mutants. Mutants very low in the wild strain and undetectable in were derived from B. flavum No. 2247 cells grown in the mutant, it was unlikely that the enzyme Medium 7 through treatment with 1 mg/ml of TV-methyl- served to produce pyruvate during growth on TV'-nitro-TV-nitrosoguanidine at 30°C for 15 min, and se- glucose. PEP: glucose PTSs, catalyzing the lected from colonies grown on Medium 7-agar plates by the replica-plating melthod. After incubation for 24 or 48 hr at formation of G6Pand pyruvate from glucose 30°C, colonies which grew on Medium 41 but not on and PEP, are widely distributed among bac- Medium 41 containing ribose instead of glucose were teria which metabolize glucose through the picked up. From among them, mutants which required Embden-Meyerhof-Parnas pathway.15) The pyruvate for growth on the ribose mediumwere further PTSactivity was assayed using toluenized cells selected. Their PK activities were assayed using CTAB- treated cells and confirmed using cell-free extracts. by measuring the glucose-dependent pyruvate

Table I. Specific Activities of Enzymes RESULTS Catalyzing Pyruvate Formation in the B. flavum Wild Strain'and Pyruvate Enzymescatalyzing the formation ofpyruvate KlNASE-DEFICIENT MUTANT Strain No. 703) was isolated as a revertant The experimental conditions were given under with a normal level of homoserine dehydro- Materials and Methods, except that the cells for the genase from strain 1-231 with a low level of pyruvate kinase assay were cultured in Medium 34 the enzyme and lacking PK, which was derived supplemented with 300/zg/liter of biotin, and that the from a B.flavum mutant, 15-8, and selected as reaction mixture for the FBP-dependent pyruvate kinase to resistance to S-(2-aminoethyl)-L-cysteine assay contained 1 mMFBP. and lysine-productivity.12'13) The PK-lacking S p e cific a c tiv ity 2 2 4 7 N o. 7 0 (n m o l/ m i n/ m g p ro t e in ) (W ild ) (P R " ) mutant, No. 70, was found to grow well on glucose (see Table III), suggesting that other Py ru va t e ki n as e 8 0 9 2 Ox al o ac et a te de ca r bo x yl as e 2 7 0 2 2 reactions might also contribute to the pyruvate M a lic en z y m e 6.3 0 .0 formation during growth on glucose. Possible In d u c i b le F B P - d e pe n d e n t PK 0 0 enzymes catalyzing the reactions were exam- PE P : g l uc o s e PT S * 4 4 2 0 ined; inducible fructose 1,6-bisphosphate (FBP)-dependent PK , oxaloacetate nmol/min/cells giving 1 mg protein of cell free decarboxylase, malic enzyme, and PEP : sugar extract. 132 M. Mori and I. Shiio

Table II. Phosphoenolpyruvate: Glucose Table III. Growth on Various Sugars and Phosphotransferase System in B. flavum the Effects of Pyruvate on the Growthof No. 2247 B. flavum No. 2247 and a Pyruvate Kinase- The composition of the reaction mixture was given deficient Mutant, No. 70 under Materials and Methods. G6P and F6P were The growth response was observed after 48 hr culti- determined by the method of Lang and Michal.19) vation at 30°C on Medium41-agar plates containing the R e ac tio n tim e (m in ) indicated sugars instead of glucose. The concentration of pyruvate added was 5 g/liter. 3 0 G ro w th

P y ru v a te fo rm a tio n (ju m o l/m l) C a r b o n so u rc e N o . 2 2 4 7 N o . 7 0 C o m p le te (1) 0 .3 8 2 .10 -G lu co se (2 ) 0 .0 6 4 . 0 .5 2 -M g 2 + (3) 0 .0 2 8 0 .2 4 N o n e N o n e p y r u v a t e p y r u v a t e -E n z y m e (4 ) 0 .0 0 0 .0 0 0 )- (2 ) 0 .32 1.5 8 G lu c o se + + + + + + + + S u cro se + + + + G 6 P f o r m at i o n (w m o l / ml ) F ru cto se 4- + + + ( l ) - ( 2 ) 0 . 2 2 0 . 6 2 M a lto se + 4 - G lu c o n a te + + - + + + G 6 P + F 6 P f o r m at i o n ( i / mo l / m l) R ib o se + + + + 0 )- (2 ) 0 .2 8 0 .7 6 M a n n ito l + + M a n n o se _ + _ + Ga lac to s e L a c to se 1.01 1 S o rb ito l g A d B G ly ce ro l G lu c o se 6 -p h o sp h a te N o n e +

-, no growth; +, slight growth; + +, normal growth.

0 0.04 0.08 0 20 40 60 TOLUENIZED CELL SUSPENSION REACTION TIME (MINI (ML! Fig. 1. Glucose-dependent Pyruvate Formation in B. 30 min reaction, suggesting that the G6Pform- flavum No. 2247. ed was consumedto yield metabolites other Pyruvate formed was determined according to the PTS than F6P. Washedtoluenized cells were stabi- assay methods described under Materials and Methods, lized by polyethyleneglycol and dithiothreitol except that the enzyme (toluenized cell suspension) volume added to the cell suspension during their prep- used for the assay (A) and reaction time (B) were as indicated in the figures. aration. A time-lag arose at the initial step of the reaction on the omission of preincubation of the reaction mixture without glucose. Under formation from PEP. Table II shows that the standard conditions, the plots of pyruvate pyruvate was produced from PEP in the pres- formed against cell amount and those against ence of glucose and Mg2+,a reported essen- reaction time (Fig. 1) were linear. Thus, the tial co factor. The formation of the other pyruvate formation represents PTS activity. product, G6P, after 5min reaction was only The activity was found in both the wild and 60% of the pyruvate formation. The total mutant strains, as shown in Table I. formation of G6P and fructose 6-phosphate (F6P), to which it was converted via the isom- Growthresponse to various sugars ofa pyruvate erase reaction, corresponded to the pyruvate kinase-deficient mutan t formation. This coincidence was not seen after If only the sugar-specific PTS substantially Pyruvate Formation and Sugar Metabolism in B. flavum 133

serves to produce pyruvate besides PK, the also detected in cells grown on various sugars PK-lacking mutant would not grow on sugars other than the substrate sugars, as shown in which were not catabolized by the PTS. Table Table IV. The cells grown on ribose or man- III shows that whereas a PK-deficient mutant, nitol had higher glucose PTS activities. This No. 70, grew well on glucose or fructose as the may be due to the difference in the growth wild strain did, it did not grow on gluconate or phase at which the cells were harvested, since ribose, but did grow on them in the presence of B. flavum grew slowly on ribose and very pyruvate. These results suggest that only PK is slowly on mannitol. The ratio of the activity responsible for the pyruvate formation during on glucose to that on fructose varied among growth on gluconate or ribose, and that other the cells grown on various carbon sources, reactions mayalso serve to produce pyruvate suggesting that two or more PTSs might func- during growth on glucose or fructose. Only the tion. High fructose PTS activities were observ- PTS is known to cause such sugar-specific ed in cells grown on fructose, mannitol and formation of pyruvate.15) sucrose. Thus, the operation of both an in- ducible fructose-specific PTS and a consti- Properties of the phosphoenolpyruvate : sugar tutive PTS with broad substrate specificities, phospho system acting on glucose and fructose, was assumed, PTS activities, with respect to various the latter of which alone was thought to sugars, were examined in cells grown on the function in the glucose-grown cells. On the substrate sugars, because the system was often basis of this assumption, B/A (A, PTS activity inducible.15) The activities for glucose, fruc- for glucose; B, that for fructose) in glucose- tose, ribose, gluconate, mannitol, sucrose and grown cells (=Bg/Ag) was thought to be the maltose in cells grownon the respective sub- ratio of the constitutive PTSactivities. In cells strate sugars were 31, 58, 7, 1, 3, 5 and grown on other sugars, B should be the sum of 4 nmol/min/20 /d cell suspension, respectively. the inducible fructose PTS activity and the Thus, the enzymewasfound to act on fructose, constitutive PTS activity for fructose. The in addition to glucose. These activities were latter activity should be obtained as

Table IV. Effects of Growth Carbon Sources on Table V. Sugar Specificities of the Phospho the Glucose- and Fructose Phosphotransferase transferase System in B. flavum No. 2247 Cells System in B. flavum No. 2247 Grownon Glucose or Fructose The experimental conditions were given under in R elative activity * Materials and Methods. Su bstrate 'Tnducible fructose PTS" was calculated as described Growth carbon source in the text. Bg/Ag (=0.48) was obtained from an average (10 him ) of the data in this table and Table V. G lucose F ructose P T S a c tiv ity G luco se 100 100 ( nmo l/ min /20 [ A of cel l sus pe nsi on) F ructose 39 127 C a rb o n so u rce 43 44 fo r g ro w th " In d u c ib le M an no se 1 1 G lu c o se (A ) F ru c to se (B ) fru c to se G alactose 6 4 P T S " L acto se G lycerol 1 0 3 1 G lu c o se 3 8 2 2 Sorbitol 8 6 F ru cto se 4 5 6 0 3 8 X ylo se 80 99 S u cro se 4 4 56 3 5 2-D eoxyglucose G lu cosam ine 92 117 M a n n ito 1 10 7 86 3 4 TV-A cetylg lu cosam ine 5 4 R ib o se 9 5 4 9 G l u c o n a t e 3 4 2 9 1 3 M a lto se 4 2 2 3 Relative activities for glucose as a substrate were taken as 100. 134 M. Mori and I. Shiio

AxBg/Ag. Thus, the inducible fructose- deficient mutant suggested the following re- specific PTS activity was calculated according lationship between sugar metabolism and pyr- to the following equation, as shownin Table uvate formation: (1) The PTSs indeed served IV. to form pyruvate in addition to PK, when B. flavum grew on glucose or fructose, which Inducible fructose PTS =B-A x Bg/Ag seemed to be catabolized mainly via the PTS A reasonable result was obtained that only reaction. Thus, PK-lacking mutants grow on cells grown on fructose, sucrose and mannitol, the sugars. (2) The PTS would also contribute which gave fructose, a possible inducer, ex- to the formation of pyruvate during growth on hibited significant activities. An inducible sucrose, maltose or mannitol, since they seem- fructose-specific PTS has been found to be ed to be catabolized through the PTSs after widely distributed in such as E. coli.15) conversion to glucose and/or fructose. PK- The sugar specificities of the PTS were deficient mutants, therefore, grow on these examined using glucose-grown cells. Table V sugars. (3) The PTS would not operate, when shows that the PTS acted on mannose, 2- cells grew on ribose or gluconate, because they deoxyglucose and glucosamine, in addition to neither acted as substrates for the PTSs nor glucose and fructose. It is interesting that the would yield glucose or fructose. Thus, PK- system operated in the phosphorylation of defective mutants are unable to grow on them. mannose which was hardly utilized by B. These suggestions were confirmed by the fol- flavum as a carbon source for growth. On the lowing determination of the possible enzyme other hand, the specificities of fructose-grown activities for catabolism of the respective cells were similar to those of glucose-grown sugars. cells, except that the activity on fructose was The presence of glucokinase catalyzing specifically high. This also supports the pres- ATP-dependent phosphorylation of glucose ence of an inducible fructose-specific PTS in into G6P in B. flavum had been reported addition to the constitutive PTS. previously,16) and this was confirmed, as shown in Table VI. Glucose-grown cells, how- Enzymes for sugar metabolism ever, exhibited much lower glucokinase ac- Studies on the PTSs ofB.flavum and on the tivity than PTS activity, and the affinity of the growth response to various sugars of a PK- former enzyme for glucose was relatively lower

Table VI. Enzyme Activities Related to Sugar Metabolism in B. flavum No. 2247 The experimental conditions were given under Materials and Methods. S u b stra te (h im ) P T S * K in a se D e h y d ro g e n a se H y d ro la se P h o sp h o ry la se ( = Ca r bo n s o u rc e ) (n m ol / m in / mg pr o t ei n )

G l u c o s e 1 0 4 4 2. 0 10 0 2 .7 F r uc t o s e 1 0 6 8 1. 0 10 0 1. 7 R ib o se 10 8 2 0 7 G l u c o n a t e 1 0 1 2 5 1 M a n n ito 1 10 3 0 2 4 3 S u c r o s e 1 0 6 10 0 0 30 0 0 M a l t o s e 1 0 5 10 0 0 16 3 0

nmol/min/cells giving 1 mg protein of cell free extract. -, not determined. Pyruvate Formation and Sugar Metabolism in B. flavum 135 than that of the latter, which was saturated by conate formation. Amongpossible mannitol- glucose at a concentration less than 10mM. catabolic enzymes, mannitol kinase and man- activity was barely detected in nitol dehydrogenase, only the latter enzyme cells grown on fructose. Ribose- and was showed high activity in B. flavum. It was gluconate-grown cells had high ribokinase and specific for NAD.The optimum pH for fruc- gluconokinase activities, respectively. The lat- tose formation was 10.0 and that for the ter activity was confirmed stoichiometrically reverse reaction, 7.0. Whereas kinase or p.hos- by measuring both ADPand 6-phosphoglu- phorylase activities for sucrose and maltose

Table VII. Effects of Growth Carbon Sources on Sugar-Metabolic Enzymes in B.flavum No. 2247 Cells were cultured in Medium13 containing 20g/liter of a sugar or lOg/liter each of two sugars as indicated instead of glucose. M a n n ito l C a rb o n so u rc e R ib o k in a se G lu c o n o k in a se d eh yd ro g e n a se M a lta se In v erta se ( n mo l /m i n /m g pr o te i n )

G lu c o se 16 3 4 5 4 2 7 8 R ib o se 20 7 3 2 1 2 R ib os e + g lu co se 14 1 G lu c o n a te 39 2 5 1 G lu co n at e + gl uc os e 2 0 1 M a n n ito l 2 4 3 M an ni t ol + g lu c os e 15 M a lto se 16 3 3 6 8 S u cro se 4 7 3 0 0

-, not determined.

Table VIII. Pyruvate Kinase Mutants Derived from B. flavum No. 2247 The bacterial strains were cultured at 30°C on agar plates of Medium41 or the latter containing 20g of ribose or 20g of ribose and 5 g pyruvate per liter instead of glucose. Growth was observed after 48 hr. G r o w t h P y r u v a t e k i n a s e S tr a i n G lu c o s e R i b o s e R i b o s e + p y r u v a t e C T A B c e l l E x t r a c t s *

N o . 2 2 4 7 + + + + + + 1 0 0 % 6 8 9 N o . 7 0 + + . + + 0 0

P - 1 0 + + + 5 2 2 7 + + + + 3 2 3 9 + + + + 0 0 4 7 4 - + + + 2 0 2 0 7 + + + + 2 5 2 1 3 + + + 2 2 0 + + + + 5 6 2 2 6 + + + + 1 0 1 2 3 3 + + + + 1 0 0 2 3 4 4 - + + -+ 1 4 2 3 6 + 4 - + 4 0 9 7 2 5 8 + 4 - + + 9 4

nmol/min/mg protein. -, not grownon the mediumfor enzymepreparation. 136 M. Mori and I. Shiio were undetectable, the activities of hydrolases, mation of pyruvate from PEP. Glucose and i.e., invertase and maltase, were high. fructose were found to be PTS substrates Regulation of these enzymes by growth car- among the sugars tested on which B. flavum bon sources was investigated. Table VII shows grew. There would be two PTSs in B. flavum, that ribokinase, gluconokinase and mannitol the constitutive PTS which exhibited broad dehydrogenase were induced specifically by the substrate specificity, acting upon glucose, fruc- respective substrate sugars, that mannitol de- tose, mannose, 2-deoxyglucose and glucos- hydrogenase was also repressed by glucose, amine, and the inducible fructose-specific and that maltase was inducible whereas in- PTS. They are similar to the mannosePTSand vertase was constitutive. Cells grown on mal- fructose PTS in E. coll, respectively. tose showed slightly higher invertase activity, PK-defective mutants grew on sugars which suggesting the possibility that the induced were thought to be phosphorylated by or maltase besides invertase served to form glu- catabolized through the PTS but did not grow cose fromsucrose. specifically on ribose or gluconate, on which the PTSdid not operate directly or indirectly, Direct derivation of PK-deficient mutants from suggesting that only PKand PTS contributed the wild strain to the pyruvate formation during the growth We tried to derive PK-defective mutants by on sugars. In addition, oxaloacetate decar- means of single mutation from the wild strain boxylase and malic enzyme were found not to in order to confirm the essential role of PKin serve to supply pyruvate under such con- the growth on ribose, because the previous ditions. An outline of the relationship between isolation of a PK-lacking mutant, No. 70, had pyruvate formation and sugar metabolism in been fairly complicated. As shown in Table B.flavum is given in Fig. 2. VIII, twelve mutants unable to growon ribose The presence of glucokinase catalyzing the in the absence of pyruvate were isolated. ATP-dependent phosphorylation of glucose Among them, three were defective in PK and MANNITOL SUCROSE five had low PK activity. All of the three PK- MALTOSE \A/FRUCTOSE GLUCOSE lacking mutants, P-39, P-47 and P-220, were T GLUCOSE 6-P also unable to grow on gluconate. The oxa- FRUCTOSEP j loacetate decarboxylase activities of strains P- \ FRUCTOSE6P 47 and P-220 were 365 and 28nmol/min/mg DHAP^ protein, whereastheir malic enzymeactivities =S- GAP t were 25.5 and 4.6nmol/min/mg protein, re- Pip Ml' spectively. Thus, it was found that even if PYRUVATE t oxaloacetate decarboxylase and malic enzyme CO2 GLUCONATE were present at the normal levels, they could I" not serve to produce sufficient pyruvate for 6-P GLUCONATE growth on ribose and gluconate. RIBULOSE 5 P i \ XYLUROSE5-1 V æf \ / DISCUSSION FRUCTOSE1,6P2 \ / >^-RIBOSE 5-P-yRIBOSE The presence of the PTS was demonstrated Fig. 2. Sugar Metabolism and Pyruvate Formation in in B.flavum. This is consistent with the general B. flavum. concept as to the distribution of the PTS in 1, PEP:sugar PTS; 2, ribokinase; 3, gluconokinase; 4, maltase; 5, invertase; 6, mannitol dehydrogenase; 7, bacteria,15) since B. ftavum is a facultative pyruvate kinase. anaerobe and catabolizes glucose mainly Abbreviations: P, phosphate; DHAP,dihydroxyacetone through the Embden-Meyerhof pathway.16'17* phosphate; GAP, glyceraldehyde 3-phosphate; PEP, The PTS catalyzes the sugar-dependent for- phosphoenolpyruvate. Pyruvate Formation and Sugar Metabolism in B. flavum 137 had been reported previously.16* In the present occur. Gluconate is thought to be catabolized study, the PTS was found to exhibit approx- through the pentose phosphate pathway, imately 20-times higher activity than gluco- whose presence in this organism has been kinase, suggesting that glucose was phospho- proved by tracer-experiments17) on CO2and rylated by the PTS at the expense of PEP pyruvate formation from 14C-glucose. The rather than by ATP-dependent kinase. Thus, PTSin B.flavum was unable to act on ribose. one of the two molecules of PEP yielded from Therefore, PK was essential for growth on it. one molecule of glucose would be consumed On the other hand, inducible ribokinase was for the phosphorylation of glucose to yield present that catalyzed the phosphorylation of pyruvate. Moreover, a part of the other one ribose. Thus, it appears to be catabolized via would also yield pyruvate via the PK reaction. the kinase reaction and the pentose phosphate This explains the increases in the productivity pathway. Since mannitol can yield fructose of aspartate3) and lysine13) caused by a PK through the mannitol dehydrogenase reaction, deficiency, which saves PEP, because they are which seems to be catabolized through the produced from PEP, and from equal amounts PTS reaction, PKis not necessary for growth of PEP and pyruvate, respectively. Only one on it. Sucrose and maltose appear to be hy- fortuitously isolated PK-lacking mutant had drolysed by constitutive invertase and induc- previously been available to improve these ible maltase into glucose and/or fructose, re- amino acid productivities by means of a PK spectively, which are thought to be phospho- deficiency. Therefore, it is industrially signifi- rylated through the PTS. Thus, PK is not in- cant that the easy selection of PK-deficient dispensable for growth on these sugars. mutants from any parent strains has become Acknowledgments. The authors are indebted to Dr. Y. possible. Wehave already obtained improved Komachiya and Dr. R. Tsugawa of their laboratories for lysine-producing mutants with a PKdeficiency the encouragement during this study. by means of the present method. The details will be given in a separate paper. REFERENCES Pyruvate formation in E. coli is thought to be catalyzed by constitutive AMP-activated 1) M. Mori and I. Shiio, J. Biochem., 98, 1621 (1985). 2) H. Ozaki and I. Shiio, /. Biochem., 66, 297 (1969). PK, an inducible FBP-dependent one, the 3) M. Mori and I. Shiio, Agric. Biol. Chem., 48, 1189 PTS, and the methylglyoxal by-pass under (1984). glycolytic conditions. Since the methylglyoxal 4) S. Sugimoto and I. Shiio, Agric. Biol. Chem., 46, 2711 by-pass can only function to a very slight (1982). extent for pyruvate formation, PK and the 5) I. Shiio and K. Ujigawa, J. Biochem., 84, 647 (1978). 6) I. Shiio, H. Ozaki and M. Mori, Agric. Biol. Chem., PTScontribute to most of the formation,18) as 46, 493 (1982). in B. flavum. Because of the presence of PK 7) A. H. Romano, J. D. Trifone and M. Brustolon, J. , however, it would be hard to isolate BacterioL, 139, 93 (1979). PK-lacking mutants as mutants unable to 8) I. Shiio, S. Otsuka and M. Takahashi, J. Biochem., grow on non-PTS sugars which did not yield 51, 56 (1962). PTS sugars. 9) R. L. Anderson and M. Y. Kamel, "Methods in Enzymology," Vol. IX, ed. by W. A. Wood, As to the metabolism of sugars other than Academic Press Inc., New York, 1966, p. 388. glucose, gluconokinase was specifically in- 10) J. J. Mieyal, "Methods in Enzymology," Vol. duced when B. flavum grew on gluconate. XXVIII, ed. by V. Ginsburg, Academic Press Inc., Thus, gluconate seems to be catabolized New York, 1972, p. 935. ll) A. Goldstein and J. O. Lampen, "Methods in through 6-phosphogluconate. Since PK was Enzymology," Vol. XLII, ed. by W. A. Wood, essential for the growth on gluconate, no sig- Academic Press Inc., New York, 1975, p. 504. nificant operation of the Entner-Doudoroff 12) I. Shiio, H. Ozaki and K. Ujigawa-Takeda, Agric. pathway, where one molecule of pyruvate is Biol. Chem., 46, 101 (1982). formed from one molecule of gluconate, would 13) H. Ozaki and I. Shiio, Agric. Biol. Chem., 47, 1569 138 M. Mori and I. Shiio

(1983). I. Shiio, S. Otsuka and T. Tsunoda, J. Biochem., 47, M. Malcovati and H. L. Kornberg, Biochim. 414 (1960). Biophys. Acta, 178, 420 (1969). A. G. Pertierra and R. A. Cooper, J. BacterioL, 129, H. M. Saier, Jr., Bacteriological Reviews, 41, 856 1208 (1977). (1977). G. Lang and G. Michal, ''Methods of Enzymatic I. Shiio, S. Otsuka and T. Tsunoda, J. Biochem., 46, Analysis," Vol. 3, ed. by H. U. Bergmeyer, Academic 1303 (1959). Press Inc., New York, 1974, p. 1238.