APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1986, p. 637-643 Vol. 52, No. 40099-2240/86/100637-07$02.00/0Copyright X 1986, American Society for Microbiology

Production of L-Phenylalanine from Starch by Analog-ResistantMutants of Bacillus polymyxat

KALIDAS SHETTY, DON L. CRAWFORD,* AND ANTHONY L. POMETTO III

Department of Bacteriology and Biochemistry, Idaho Agricultural Experiment Station, University of Idaho,Moscow, Idaho 83843

Received 2 December 1985/Accepted 17 June 1986

p-Fluorophenylalanine-resistant mutants of starch-degrading Bacillus polymyxa ATCC 842, generated byethyl methanesulfonate mutagenesis followed by incubation with caffeine, overproduced small amounts ofL-phenylalanine (L-phe) from starch. A B-2-thienylalanine-resistant mutant (BTR-7) derived from p-fluorophenylalanine mutant (C-4000 FPR-4) and resistant to both p-fluorophenylalanine and 1-2-thienylalanineproduced 0.5 g of L-phe and 0.15 g of L-tyrosine per liter from 10 g of starch per liter when growing in aminimal medium. trans-Cinnamic acid (CA) was also excreted by both mutants, indicating the possibility ofL-phenylalanine ammonia-lyase-induced deamination of L-phe to CA. The amount of L-phe-derived CAdetected in BTR-7 was less compared with mutant C-4000 FPR-4. CA production was induced in the parent onlywhen L-phe was used as a sole nitrogen source. Time of CA production in the two mutants could be delayedby addition of other nitrogen sources, an indication of possible L-phenylalanine ammonia-lyase inhibition orrepression. The presence of L-phenylalanine ammonia-lyase in B. polymyxa mutant C-4000 FPR-4 wasconfirmed by assays of cell-free extracts from cells grown in starch minimal medium containing L-phe as thesole nitrogen source. Preliminary studies of the regulation of deoxy-D-arabino-heptulosonate-7-phosphatesynthase and prephenate dehydratase in the wild-type strain showed that deoxy-D-arabino-heptulosonate-7-phosphate synthase was subject to feedback inhibition by L-phe, L-tyrosine, and L-tryptophan. Inhibition byeach amino acid was to a similar extent singly or in combination at a 0.5 mM level of each amino acid.Prephenate dehydratase was feedback inhibited by L-phe, but not by L-tyrosine or L-tryptophan or both. In thedouble analog-resistant mutant BTR-7, deoxy-D-arabino-heptulosonate-7-phosphate synthase had specificactivity similar to that in the wild type, and the enzyme was still subject to feedback inhibition. However,prephenate dehydratase had increased specific activity and it was also insensitive to feedback inhibition byL-phe. The overproduction of aromatic amino acids by BTR-7 was thought to be due, at least in part, toderegulation of feedback inhibition of prephenate dehydratase. Chorismate mutase was not subject to feedbackinhibition in the wild type and was unaffected in the mutant.

Starches are an abundant, underutilized waste generatedin large amounts by the potato processing industry (14).Various bioconversion processes have been developed toutilize these wastes for the production of single-cell proteinand ethanol (1, 9, 11). This paper reports on the microbialproduction of L-phenylalanine (L-phe) from starch by L-pheanalog-resistant mutants of the starch-degrading bacteriumBacillus polymyxa. Amino acid analog-resistant bacterialmutants have been utilized previously only for microbialproduction of aromatic amino acids from substrates such asglucose, sucrose, and methanol (8, 26, 31).

In analog-resistant L-phe-overproducing bacterial mu-tants, there is typically genetic removal of feedback controlof the aromatic amino acid biosynthetic pathway (7). m-Fluorophenylalanine-resistant mutants of Brevibacteriumflavum, for example, had a feedback-insensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase andoverproduced L-phe and L-tyrosine (L-tyr) in equimolarratios, but did not overproduce L-tryptophan (L-trp). Fur-thermore, mutants of B. flavum having prephenatedehydratse insensitive to L-phe inhibition produced onlyL-phe (29). A Bacillus subtilis mutant resistant to 1-2-thienylalanine (Tala) and having a prephenate dehydrase

* Corresponding author.t Paper 85513 of the Idaho Agricultural Experiment Station.

insensitive to inhibition by L-phe excreted a small quantity ofL-phe (20).

In the present work, B. polymyxa ATCC 842 wasmutagenized with ethyl methanesulfonate (EMS), and themutagenized cells were then post-incubated with caffeinebefore being plated onto agar media containing specificL-phe analogs. Several p-fluorophenylalanine (pFP)-resis-tant mutants and pFP/Tala double resistant mutants wereisolated. Overproduction of L-phe by selected mutants wasexamined by high-pressure liquid chromatography (HPLC).Production of L-phe was limited by an inducible L-phenyl-alanine ammonia-lyase (PAL) which mediated deaminationof L-phe to trans-cinnamic acid (CA). Some preliminaryenzymatic studies of one L-phe- and L-tyr-overproducing,analog-resistant mutant showed that it has a partially dereg-ulated biosynthetic pathway.

MATERIALS AND METHODSMicroorganism and media. B. polymyxa ATCC 842 was

obtained from the American Type Culture Collection(Rockville, Md.), maintained on starch minimal medium(SMM) agar slants, and stored at 4°C. The composition ofthe SMM used for growth of the parent culture and theisolated mutants (per liter) was as follows: 10.0 g of starch,2.0 g of (NH42504, 5.0 g of Na2HPO4, 3.0 g of KH2PO4, 0.2

637

on April 13, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

g of MgSO4. 7H20, 0.2 g of NaCl, 0.5 g of CaCl2 2H20,and 1 ml of trace elements (19), pH 7.1. In media whereL-phe was used, the concentration of L-phe was 1.0 g/liter.Cultures were grown at 37°C under constant aeration asshake flask cultures (100 rpm).Mutagenesis and isolation of analog-resistant mutants. B.

polymyxa ATCC 842 was incubated shaking at 37°C in 100 mlof SMM to mid-log phase (36 h). Cells were harvested bycentrifugation of 10-ml samples in sterile tubes at 3,000 x gfor 20 min. The pellet was suspended in 10 ml of SMM, and0.4 ml of anhydrous ethyl methanesulfonate (EMS) wasadded under aseptic conditions. This suspension was shakenat 37°C for 1 h, which resulted in a 95% kill as shown by killcurves determined previously (not shown). The survivingcells were washed three times in sterile 0.05 M phosphatebuffer (pH 7.0), transferred into 100 ml of SMM, andincubated with shaking at 37°C for 36 h. A second EMStreatment was then carried out as above at one-half the dose(0.2 ml) to give a 95% kill in 90 min. The surviving cells wereaseptically centrifuged, washed once in phosphate buffer,and transferred to 10 ml of SMM containing 5 mg of caffeineper mrl. These cells were incubated at 37°C for 4 to 5 h withconstant aerobic shaking in the dark. The cells were thenaseptically centrifuged, washed three times in phosphatebuffer, and suspended in 10 ml of sterile distilled water.Appropriately diluted cells were then plated on SMM agarsupplemented with 4 mg of pFP, 100 ,ug of Tala, or 0.5 to 1.0mg of other amino acid analog per ml, and incubated at 37°C.Resistant colonies, which grew slowly, were isolated after 5to 7 days of incubation. The Tala/pFP-resistant mutantswere derived from mutant C-4000 FPR-4, a pFP-resistantmutant isolated by the procedure summarized above. Theconcentration of Tala used in all selections was 100 ,ug/ml.Growth curve, amylase activity, and total carbohydrate

determinations. B. polymxa ATCC 842 was incubated byshaking at 37°C in 100 ml of SMM, and samples wereremoved periodically. Cell growth was measured byturbidometric assay of samples at 600 nm. For amylaseassays and total carbohydrate determinations, culture brothsamples from the above medium were centrifuged at 10,000x g for 15 min. Extracellular amylase activity in brothsamples was determined by following increases in the con-centration of reducing sugar (as maltose) over time inreaction mixtures containing 1-ml samples of culture super-natant incubated with starch, using the 3,5-dinitrosalicylicacid method of Bernfeld (2). The micromoles of reducingsugar liberated by amylase were calculated from a maltosestandard curve. Activities were based upon initial linearreaction rates, and 1 U of amylase activity was defined as theamount of enzyme liberating 1 ,umol of reducing sugar (asmaltose) per min at 25°C and pH 6.9. For determination oftotal carbohydrate, 1-ml samples of culture supernatant wereassayed by the anthrone method (16). In this assay, carbo-hydrate depletion was estimated by measuring changes inA540, based on a standard curve prepared with starch. Totalcarbohydrate in culture supernatant samples was expressedas grams of carbohydrate per 100 ml of medium.

Cross-feeding assay for selection of L-phe-overproducingmutants. Mutants resistant to pFP and Tala were streakedacross the center of a petri plate containing SMM agarsupplemented with 1 mg of pFP per ml and were incubated at37°C for 72 h. Then the parent strain was cross-streakedclose to the central growth zone of the mutants. If the parentstrain grew on the medium containing the analog, the mu-tants were assumed either to overproduce L-phe or todegrade the pFP, thereby supporting the growth of the

parent. The parent strain alone failed to grow on the analog-supplemented medium.HPLC, Selected cross-feeding-positive mutants were

grown in SMM. Supernatants were sampled at 24, 48, 72,and 96 h. The samples were mixed with methanol (1:1) andfiltered through a 0.45-,um filter (Gelman Sciences, Inc., AnnArbor, Mich.). Then, 50 ,l of filtrate was injected into aHewlett-Packard (Bellevue, Wash.) 1090A HPLC equippedwith a 1040A diode array detector and a 5-1ul sample-applying loop. The solvent used was a gradient of acidicwater, pH 3.25 (H2SO4), and methanol at an initial concen-tration of 20% methanol. The methanol concentration washeld at 20% for 2 min, increased to 60% over the next 2 min,and then held at 60% for 2 min, after which it was returnedto 20% over the last 2 min (total run time, 8 min). Amicrobore reverse-phase column (100 by 2.1-mm insidediameter; Hewlett-Packard) of Hypersil ODS with a 5-,umparticle diameter was used with a flow rate of 0.4 ml/min anda column temperature of 40°C. During each run, a chromato-gram was recorded at 258 nm, and the UV absorbancespectrum (250 to 350 nm) of each chromatographic peak wasrecorded at its front, apex, and backside. Under theseconditions the retention time of CA was 5.55 min and thepeak exhibited a sharp absorption maximnvm at 276 nm,whereas L-phe had a retention time of 1.35 min, with a sharpabsorption maximum at 258 nm. Retention times did varyslightly. This was corrected for by periodic chromatographyof standards and by examination of UV absorption spectra.A standard curve for L-phe was prepared by plotting aknown concentration of L-phe versus peak area units, re-corded with a Hewlett-Packard 3390 integrator. A standardcurve for L-tyr was prepared similarly. L-Tyr exhibited aretention time and absorption maximum of 1.05 min and 274nm, respectively. Urocanic acid, another potential product,exhibited a retention time of 4.25 min and an absorptionmaximum of 268 nm.PAL assay. B. polymyxa ATCC 842 and selected mutants

were incubated aerobically at 37°C for 2 days, and cells werethen harvested by centrifugation at 10,000 x g for 15 min.The cells were suspended in 0.05 M Tris-hydrochloridebuffer (pH 8.5), sonically disrupted, and then centrifuged at27,000 x g for 15 min (30). PAL activity was determined byusing the supernatant as the cell-free extract. The assayinvolved incubating 0.2 ml of extract in 0.8 ml of Tris-hydrochloride buffered 15 mM L-phe for 1 h at 37°C. Thereaction was terminated by addition of methanol (1:1). Thenthe solution was filtered and examined by HPLC as above.However, the solvent system used was acidic water with40% methanol maintained isocratically. The retention timesfor L-phe and CA were 0.98 and 2.3 min, respectively.GLC. Culture supernatants from the various experiments

were acidified to pH 1 to 2, extracted twice with ether andonce with ethyl acetate; the combined organic phases weredewatered with sodium sulfate and evaporated to dryness.Then, preweighed 2- to 3-mg amounts of extracted com-pounds from each supernatant were converted to tri-methylsilyl derivatives by addition of 100 ,u of p-dioxanecontaining an internal standard (0.1 mg of 3,4-dimeth-ylbenzaldehyde), 10 ,ul of pyridine, and 50 RId of N,O-bis-(trimethylsilyl)acetamide (6). Each sample was held at 35°Cfor 2 h prior to injection. Gas-liquid chromatography (GLC)was performed on a Hewlett-Packard 5890 gas chromato-graph with a flame ionization detector and RSL-150 capillarycolumn (30 m by 0.25 mm) (Alltech Associates, Inc.,Deerfield, Ill.). Chromatographic conditions were as follows:oven temperature of 120°C for 2 min, followed by a 20°C/min

638 SHETTY ET AL.

on April 13, 2020 by guest

http://aem.asm

.org/D

ownloaded from

L-PHE PRODUCTION FROM STARCH BY B. POLYMYXA MUTANTS 639

gradient to 260°C, which was then held for 3 min. Theinjector temperature was 240°C and the detector temperaturewas 280°C. The retention time for CA was 6.80 min; that forurocanic acid was 9.12 min.

Biosynthetic enzyme assays. B. polymyxa ATCC 842 andmutants C4000 FPR-4 and BTR-7 were incubated aerobicallyat 37°C in SMM for various lengths of time. Cells were

harvested by centrifugation at 10,000 x g for 15 min, and thepellet was suspended in a small volume of 50 mM phosphatebuffer (pH 7.0). Cells were then disrupted by passage twicethrough a French pressure cell (Aminco Inc., Silver Spring,Md.) at 12,000 lb/in2. Disrupted cells were centrifuged at27,000 x g for 15 min, and the supernatnat was used as thesource of enzyme for the assays described below. DAHPsynthase was assayed by the procedures of Jensen andNester (10) and Srinivason and Sprinson (28). Chorismatemutase was assayed by the procedure of Patel et al. (19).End product inhibition studies involved the addition of thearomatic amino acids, singly or in combination, to reactionmixtures assayed by the procedures summarized above. Inthese studies, 0.2-ml volumes of amino acid solutions were

added to the reaction mixtures. The final concentration ofeach amino acid was 0.5 mM.

Protein determinations. Protein concentration was deter-mined by the method of Lowry et al. (15), using bovineserum albumin as the standard.

Chemicals. L-Phe, pFP, Tala, tryptophan hydroxamate,5-methyltryptophan, 5-fluorotryptophan, azaserine, 7-aza-tryptophan, 2-amino-3-phenylbutanoic acid, L-tyr, CA, N,O-bis-(trimethylsilyl)acetamide, 3,5-dinitrosalicylic acid, EMS,caffeine, erythrose-4-phosphate, phosphoenolpyruvate,chorismic acid, prephenic acid, and bovine serum albuminwere purchased from Sigma Chemical Co. (St. Louis, Mo.).HPLC-grade methanol, dioxane, pyridine, and anthronewere from Baker Chemical Co. (Phillipsburg, N.J.). Solublestarch was from Difco Chemical Co. (Detroit, Mich.). Allother chemicals were laboratory reagent grade.

RESULTS

Growth of B. polymyxa on starch. Amylase activity andcarbohydrate utilization over time by B. polymyxa were

determined for cells grown in SMM. The initial lag periodlasted approximately 4 h and was followed by 44 h oflog-phase growth. The culture reached stationary phase in 48h. Culture turbidity then remained unchanged through 72 hbefore the absorbance started falling. Increasing extracellu-lar amylase activity and cell growth closely correlatedthrough 36 h of incubation. Amylase activity peaked at 36 h(2 U of activity per ml) and then fell rapidly. Total carbohy-drate depletion was slow until near the beginning of station-ary phase. Rapid utilization of carbohydrates was observedbetween 48 and 72 h, by which time 90% of the anthrone-reactive substances had been depleted from the culturemedium.

Isolation of mutants and cross-feeding assay. The frequencyof isolation of L-phe analog-resistant mutants among muta-genesis survivors was calculated by counting the averagenumber of colonies per milliliter appearing on the SMMmedium-containing analog and dividing by the average num-

ber of colonies per milliliter appearing on SMM mediumwithout analog. Frequencies of isolations were also calcu-lated when the caffeine postincubation step was eliminatedfrom the procedure. In addition to L-phe analogs (pFP, Tala,and 2-amino-3-phenylbutanoic acid), several L-trp (trypto-phan hydroxymate, 5-methyltryptophan, 5-fluorotrypto-

TABLE 1. Frequency of L-phe and L-trp analog-resistant mutantswith and without caffeine postincubation of mutagen-treated cells

of B. polymyxa ATCC 842a

Frequency, 106

Analog EMS mutagenesis UV mutagenesis

+ Caffeine - Caffeine + Caffeine - Caffeine

L-Trp analog65-Methyltryptophan 29.0 10.8 30.0 12.05-Fluorotryptophan 28.0 9.8 24.0 18.07-Azatryptophan 14.0 6.4 11.0 8.4Tryptophan 35.0 18.0 30.0 14.0hydroxamate

Azaserinec 4.4 0.8 2.4 1.1L-Phe analog

4-Fluorophenyl- 38.0 14.0 18.0 11.0alanine

2-Amino-3-phenyl- 42.0 16.0 16.0 14.0butanoic acid

Tala 4.6 0.6 2.6 0.7a Values are averages of three replicates of colonies per milliliter appearing

on medium containing analog divided by colonies per milliliter appearing onmedium without analog. All values for + caffeine were significantly differentfrom those for - caffeine at the 0.05 level (P < 0.05) as tested by the generallinear model (18).

bConcentrations of analog in the SMM medium were as follows: 5-methyltryptophan, 1 mg/ml; 5-fluorotryptophan, 1 mg/ml; 7-azotryptophan,0.5 mg/ml; tryptophan hydroxamate, 1 mg/ml; azaserine, 0.5 mg/ml; 4-fluorophenylalanine, 1 mg/ml; 2-amino-3-phenylbutanoic acid, 1 mg/ml; Tala,50 ,ug/ml.

c Serine analog affecting L-trp pathway at level of tryptophan synthase.

phan, 7-azotryptophan) and one serine analog (azaserine)were also used, and frequencies of appearance of analog-resistant colonies were also calculated with and without thecaffeine postmutagenesis incubation step. The frequencies ofgeneration of analog-resistant B. polymyxa mutants undereach condition and with each analog are shown in Table 1.With each analog, the frequency of isolation of resistantmutants was significantly higher when the caffeine incuba-tion step was incorporated into the procedure. For example,with pFP, the frequency increased from 1.4 x 107 whencaffeine postincubation was omitted to 3.8 x 107 when it wasincluded. Similar results were observed when UV light wasutilized as mutagen instead of EMS, and similar patternswere observed for each of the eight amino acid analogsexamined.

In all experiments to isolate analog-resistant L-phe-overproducing mutants of B. polymyxa, either pFP or Talawas utilized as an analog at a concentration of 4 mglml or 100,ug/ml, and all isolations included postincubation of muta-genesis-surviving cells with caffeine as described above.Eighteen mutants resistant to pFP were isolated initially. Allwere confirmed to be B. polymyxa by morphological andphysiological comparison with the wild-type strain. Three ofthe mutants, C-4000 FPR-4, H-4000 FPR-4, and G-4000FPR-2, supported growth of the parent when it was cross-streaked on SMM agar medium containing 1 mg of pFP perml. These mutants were, therefore, assayed by HPLC foroverproduction of L-phe.HPLC analysis confirmed that mutants positive by the

cross-feeding assay were L-phe overproducers. L-Phe wasdetected in the culture supernatant of each mutant after 72 hof growth in SMM. Mutants H-4000 FPR-4, G-4000 FPR-2,and C-4000 FPR-4 produced 0.15, 0.18, and 0.25 g of L-pheper liter, respectively. After 72 h, however, apparent deam-ination of the accumulated L-phe to CA was observed. Thefirst detection of extracellular L-phe coincided with the time

VOL. 52, 1986

on April 13, 2020 by guest

http://aem.asm

.org/D

ownloaded from

640 SHETTY ET AL.

TABLE 2. L-Phe overproduction by mutants of B. polymyxaresistant to both Tala and pFPa

Mutant L-Phe CA(g/liter)

BTR_1 0.10 +BTR-2 0.14 +BTR-3 0.08 +BTR-4 0.28 +BTR-5 0.35 +BTR-6 0.22 +BTR-7 0.50 +BTR-8 0.33 +BTR_9 0.11 -BTR_10 0.16 +BTR_11 0.11 +BTR_12 0.32 +Parental C4000-FPR-4 0.25 +

a Culture supernatants were assayed by HPLC after 72 h of aerobic growthin SMM at 37'C.

of maximum depletion of total carbohydrates from themedium, and further accumulation was observed well intothe stationary phase of growth. Since mutant C-4000 FPR-4accumulated the greatest amount of L-phe (0.25 g/liter), itwas later used as parent to generate Tala-resistant mutants.HPLC analysis showed that by 96 h of incubation much of

the L-phe present in culture supernatants from mutantC-4000 FPR-4 had been replaced by the L-phe deaminationproduct CA. The presence of CA was confirmed by HPLCand GLC. This indicated the presence of PAL in B.polymyxa. C-4000 FPR-4 was therefore grown with L-phe assole nitrogen source in a medium containing starch as thesole carbon source. Under these conditions, extracellularCA was detected after 2 days of incubation. When L-phe wasprovided along with ammonium sulfate as the nitrogensource, however, CA production was delayed until day 4 or5, indicating PAL repression or inhibition by (NH4)2SO4.Furthermore, CA production was induced in the parent onlywhen L-phe was used as a sole nitrogen source. Though itcould serve as a nitrogen source, L-phe would not supportsignificant growth as a sole nitrogen and carbon source.HPLC was used for assaying PAL in crude cell-free

extracts. The PAL assay detected formation of CA afterincubation of crude cell-free extracts with L-phe for 30 min.GLC analysis confirmed the production of CA both inculture supernatants of the mutants and by cell-free extractsin the PAL assay, using C-4000 FPR-4 extracts incubatedwith L-phe.Growth, amylase activity, and carbohydrate utilization abil-

ity by mutant C-4000 FPR-4. The growth curve of C-4000FPR-4 was similar to that of the parent. However, amylaseactivity, though it followed the same pattern as the parent,peaked at a higher activity (2.42 U/ml at 36 h versus 2.0 U/mlin the parent). Again, carbohydrate utilization was not rapidprior to 48 h. Between 48 and 72 h, the rate of depletion ofcarbohydrate was equal to that seen with the parent. Theanthrone procedure was not sensitive enough to detect anyminor differences in carbohydrate depletion rates.

Isolation and characterization of Tala-resistant mutants.Tala-resistant mutants were isolated by EMS mutagenesis ofC-4000 FPR-4. Twelve mutants were isolated. All of themutants were resistant to 4 mg of pFP and 100 jig of Tala perml, and all were strongly positive in the cross-feeding growthassay.Each of the 12 mutants were also positive for L-phe

overproduction, with mutant BTR-7 being the best of the

group (Table 2). BTR-7 produced 0.5 g of L-phe per liter from10 g of starch per liter after 72 h of incubation in SMM. Thepattern of production of L-phe and deamination to CA wassimilar to that observed with C-4000 FPR-4. About half ofthese mutants produced less L-phe than did the parent,C-4000 FPR-4. It is possible that these mutants may be oneswhich diverted some of their excessive carbon flow intoother pathways, thereby draining carbon away from theL-phe pathway. BTR-7, for example, also overproduced 0.15g of L-tyr per liter. We did not examine each of the mutantsfor this possibility.Growth, amylase activity, and carbohydrate utilization by

BTR-7. Growth of mutant BTR-7 was similar to that observedfor C-4000 FPR-4, as was amylase activity at 36 h (2.52U/ml). BTR-7 exhibited a similar amylase production curvecompared with C-4000 FPR-4. Carbohydrate utilization wasrapid between 32 and 36 h, and 80% of the carbohydrate inthe medium had been depleted by 72 h.

Activities and regulatory patterns of key biosynthetic en-zymes. The specific activities ofDAHP synthase, prephenatedehydratase, and chorismate mutase were followed overtime for the parent strain ATCC 842 and for mutants C-4000FPR-4 and BTR-7. Specific activities were determined after24, 48, 72, and 96 h of growth in SMM at 37°C. DAHPsynthase, prephenate dehydratase, and chorismate mutaseeach exhibited maximal activity between 48 and 72 h in all ofthe cultures, and the time of maximum activity paralleled thetime of entry of the cells into stationary phase. Maximumspecific activities for each culture are shown in Table 3.DAHP synthase exhibited significantly higher specific activ-ities in mutant C-4000 FPR-4 than in parent ATCC 842. Thespecific activities of prephenate dehydratase was also signif-icantly higher in both mutants. The level of chorismatemutase, however, was essentially unchanged in the mutantsversus the wild-type strain.

Studies of end product inhibition ofDAHP synthase in thewild-type strain showed that the enzyme was inhibited to thesame extent by any single aromatic amino acid (L-phe, L-tyr,or L-trp), using 0.5 mM concentrations of an individualamino acid. A similar level of inhibition was observed whenequimolar combinations of L-phe, L-tyr, and L-trp (0.5 mMeach) were added to the reaction mixture. This suggests thatL-phe, L-tyr, and L-trp may occupy the same allosteric siteon the enzyme, though further study will be required todetermine if this is actually the case. Increasing amino acidconcentrations to 1.0 mM in the reaction mixtures caused noincreased inhibition in any of the combinations examined.

In the parent, prephenate dehydratase was feedback in-

TABLE 3. Peak specific activities of selected shikimate pathwayenzymes in cell-free extracts from wild-type B. polymyxa ATCC

842 and two L-phe analog-resistant mutantsa

Sp act (nmol of product formed min-' mg ofprotein-l)b

OrganismDAHP Prephenate Chorismatesynthase dehydratase mutase

Wild-type ATCC 842 5.8 2.7 3.2Mutant C-4000 FPR-4 12.4c 4.8c 3.2Mutant BTR-7 6.2 5.4C 3.3

a Cell-free extracts were prepared after 48 to 72 h of shaking incubation ofcultures grown in SMM at 37°C.bAt 37°C under the specific assay conditions described for each enzyme.

Values are averages of three determinations.c Value was significantly different from all other values in its column but not

from each other at the 0.05 (P < 0.05) level as tested by the general linearmodel (18).

APPL. ENVIRON. MICROBIOL.

on April 13, 2020 by guest

http://aem.asm

.org/D

ownloaded from

L-PHE PRODUCTION FROM STARCH BY B. POLYMYXA MUTANTS 641

hibited by 0.5 mM L-phe, but not by L-tyr or L-trp. Variousequimolar (0.5 mM each) combinations of the amino acids(L-phe plus L-trp; L-phe plus L-tyr; L-tyr plus L-trp; L-pheplus L-tyr plus L-trp) caused no change in the level ofinhibition observed (similar extent of inhibition as seen whenL-phe only is added in the combination). When L-phe was

added at a 1.0 mM concentration, inhibition increased fur-ther (data not shown).

In mutants C-4000 FPR-4 and BTR-7 DAHP synthase was

inhibited in a pattern similar to that observed with theparent. Also, in C-4000 FPR-4 prephenate dehydratase was

still subject to feedback inhibition as in the parent (data notshown). However, in BTR-7 prephenate dehydratase was

feedback inhibition insensitive. The inhibition patterns forDAHP synthase and prephenate dehydratase in BTR-7 are

shown in Table 4.

DISCUSSION

The results show a clear pattern where caffeine post-incubation in the mutagenesis procedure led to an enhancedfrequency of recovery of L-phe and L-trp analog-resistantmutants of B. polymyza. One explanation of the mechanismfor the observed results is that of caffeine-enhanced activityof SOS repair enzymes. More active SOS repair systemcould be responsible for the enhanced isolation frequenciesfor both L-phe and L-trp analog-resistant mutants. Mutationsare caused by errors made by DNA repair systems that allowbacteria to survive with their DNA damaged (32). At highdoses of mutagens constitutive repair mechanisms such as

excision repair are overloaded (13, 27). At this stage, error-

prone SOS enzymes may be induced. SOS-like repair en-

zymes can repair damaged DNA strands in the absence ofDNA template (13, 27). Caffeine has been shown to cause an

increase in mutation frequency of isolates from Schizosac-charomyces pombe, although the increase was also depen-dent on high UV doses (4). If caffeine has a similar broadeffect on DNA processing and repair in B. polymyxa, then itsmechanism for inducing higher frequencies of L-phe- andL-trp-resistant mutants may very well be similar. Studies ofthe parent strain indicated that the optimum time for cellharvest and mutagenesis was during mid- to late log phase ofgrowth (32 to 40 h).The cross-feeding assay provided a rapid way to visualize

these L-phe-excreting mutants. From 18 pFP-resistant mu-

tants isolated, only three were positive in the cross-feedingassay. HPLC confirmed that growth of the parent strain inthe cross-feeding plate assay for these three mutants was dueto excretion of L-phe by the analog-resistant mutants, not toneutralization of the pFP by the mutants.

BTR-7, a Tala- and pFP-resistant mutant, overproduced0.5 g of L-phe and 0.15 g of L-tyr per liter in the SMM, whichcontained 10 g of starch per liter. This mutant was the bestoverproducer of all mutants examined. An unexpected ob-servation was that biosynthetic overproduction of L-phe inthese mutants was observed only after the cells were ap-proaching or had entered stationary phase. However, thiswas observed repeatedly. PAL-catalyzed conversion of L-

phe to CA could not be totally responsible for this observa-tion. However, because of L-phe's weak extinction coeffi-cient, the earlier accumulation of L-phe that probablyoccurred was not detectable by the procedures used. Inaddition, it is possible that the extracellular release ofpreviously synthesized L-phe may have occurred only afterthe cells entered stationary phase or began to lyse as theyentered death phase or both.

TABLE 4. Effect of aromatic amino acids on specific activities ofDAHP synthase and prepheante dehydratase of B. polymyxa

mutant BTR-7a

Sp act (nmol of product formedSubstrate effector(s)b min-1 mg of protein-l)c

DAHP Prephenatesynthase dehydratase

Controld 6.2e 4.5L-Phe 4.3 4.4L-Trp 4.5 4.2L-Tyr 5.1 4.5L-Phe + L-trp 4.3 4.6L-Phe + L-tyr 4.1 4.2L-Tyr + L-trp 3.6 4.1L-Tyr + L-trp + L-phe 4.4 4.4

a Cell-free extracts were prepared after 48 to 72 h of shaking incubation ofcultures grown in SMM at 37°C.

b Final concentration of each amino acid was 0.5 mM.c At 37'C under the specific assay conditions described for each enzyme.

Values are averages of three determinations.d Activity with no additions.e Value was significantly different from all other values in its column at the

0.05 level (P < 0.05) as tested by the general linear model (18).

An interesting finding was that accumulation of L-phe bythe mutants was limited by its deamination to CA in areaction mediated by PAL. This has also been seen in theyeast Rhodotorula glutinis (12, 33). Interestingly, the discov-ery of PAL in B. polymyxa is unique. To our knowledge, noreports of PAL in unicellular gram-positive bacteria haveappeared in the literature. Streptomyces verticillatus is theonly other procaryote thus far shown to have this enzyme(3).The present findings confirm that L-phe was converted to

CA by cell-free extracts prepared from cells of mutantC-4000 FPR-4 grown in SMM containing L-phe as the solenitrogen source. There is a possibility that the CA detectedmay have been contaminated by urocanic acid, a closelyrelated compound produced by deamination of the aminoacid histidine. This possiblity was eliminated since urocanicacid was not detected by HPLC or GLC, which readilydifferentiated urocanic acid from CA.The steady increase in specific activity of DAHP synthase

and prephenate dehydratase during growth of the culturesshows that these enzyme activities parallel the growth curveof B. polymyxa. Chorismate mutase specific activity, how-ever, remained constant over time. Chorismate mutase maytherefore be important in channeling of carbon to sidebranch pathways in the general sequence of reactions toeach of the aromatic amino acids. Chorismate mutase spe-cific activity was also similar between the parent and themutants. A better understanding of its regulatory role in B.polymyxa is needed. DAHP synthase specific activity in theparent strain was feedback inhibited to the same extent byany single aromatic amino acid. Similar inhibition was de-tected when L-phe, L-tyr, and L-trp were added in differentcombination. This is in contrast to the observed regulation ofDAHP synthase in R. glutinis and Escherichia coli, whoseenzymes exhibit an additive pattern of feedback inhibition,with DAHP synthase existing as three distinct isozymes (7,21, 28). In Brevibacterium flavum, DAHP synthase is syn-ergistically inhibited by L-phe and L-tyr (25). However, inBacillus subtilis DAHP synthase is a single isozyme andDAHP synthase and chorismate mutase form a bifunctionalenzyme (17).The higher specific activity of DAHP synthase in C-4000

VOL. 52, 1986

on April 13, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

FPR-4, as compared with the parent (Table 3), indicates thatthe enzyme is either partially or wholly derepressed, or elsethe increased specific activity is due to a structural mutationin the synthase gene, which in turn codes for a more activeprotein. The increased activity of DAHP synthase is proba-bly the principal reason for overproduction of L-phe by thismutant. Derepression of DAHP synthase has, for example,contributed to overproduction of L-trp by B. flavum (24, 26).In B. subtilis, a fluorotryptophan-resistant mutant with a280-fold increased level of anthranilate synthase activityexcreted L-trp (23). Mutant BTR-7 also had a specific activityfor DAHP synthase that was similar to the parent. This couldbe the result of a double mutation, since BTR-7 was derivedfrom C-4000 FPR-4 which had enhanced DAHP synthaseactivity as compared with the parent.While prephenate dehydratase also exhibited an increased

specific activity in both mutants, the prephenate dehydratasewas feedback inhibition insensitive only in mutant BTR-7. Afeedback-insensitive prephenate dehydratase in B. flavumhas previously been shown to contribute to the overproduc-tion of L-phe (29). Our results suggest that the greaterproduction of L-phe in BTR-7 versus C-4000 FPR-4 could bedue to mutations resulting in deregulation of prephenatedehydratase so that it is insensitive to feedback inhibition byL-phe. In addition, it is possible that a lower PAL activity ascompared to C-4000 FPR-4 might also promote greater L-pheaccumulation. We have not yet examined this possibility.

It is probable that the increased specific activities andderegulation of feedback inhibition of prephenate dehy-dratase are the principal reasons for overproduction of L-pheand L-tyr by mutant BTR-7. Further improvement of over-production might be accomplished by obtaining DAHPsynthase feedback-insensitive mutants and by further in-creasing the specific activities of both DAHP synthase andprephenate dehydratase in this strain. In addition, the devel-opment of auxotrophy to L-tyr and L-trp in the mutant wouldalmost certainly enhance production of L-phe. Finally, theisolation of PAL-negative mutants will be necessary to avoidcatabolic loss of L-phe through deamination to CA.

ACKNOWLEDGMENTSWe thank Lee A. Deobald for technical advice.This research was supported by the Idaho Agricultural Experi-

ment Station and by a seed grant from the University of Idaho,Office of University Research.

LITERATURE CITED

1. Admassu, W., R. A. Korus, and R. C. Heimsch. 1984. Kineticsof the amylase system of Saccharomycopsisfibuligera. EnzymeMicrobiol. Technol. 6:549-554.

2. Bernfeld, P. 1951. Enzymes of starch degradation and synthesis.p. 379. In F. F. Nord (ed.), Advances in enzmology, vol. 8.Interscience Publishers, Inc., New York.

3. Bezanson, G. J., D. Desaty, A. V. Emes, and L. C. Vining. 1970.Biosynthesis of cinnamide and detection of phenylalanine am-monia lyase in Streptomyces verticillatus. Can. J. Microbiol. 16:147-151.

4. Clark, C. H. 1973. Differential effects of caffeine in mutagentreated Schizosaccharomyces pombe. Mutat. Res. 46:243.

5. Cotton, R. G. H., and F. Gibson. 1965. The biosynthesis ofphenylalanine and tyrosine; enzymes converting chorismic acidinto prephenic acid and their relationship to prephenate dehy-drogenase. Biochim. Biophys. Acta 100:76-88.

6. Crawford, D. L., A. L. Pometto III, and R. L. Crawford. 1983.Lignin degradation by Streptomyces viridosporus: isolation andcharacterization of a new polymeric lignin degradation interme-diate. Appl. Environ. Microbiol. 45:898-904.

7. Fiske, M. J., and J. F. Kane. 1984. Regulation of phenylalaninebiosynthesis in Rhodotorula glutinis. J. Bacteriol. 160:676-681.

8. Hagino, H., and K. Nakayama. 1973. L-Tyrosine production byanalogue resistant mutants, derived from a phenylalanineauxotroph of Corynebacterium glutamicum. Agric. Biol. Chem.37:2013-2023.

9. Hendricks, B., R. A. Korus, and R. C. Heimsch. 1985. Propionicacid production by fermentation. Biotechnol. Bioeng. Symp.Ser. 15:241-245.

10. Jensen, R. A., and E. W. Nester. 1966. Regulatory enzymes ofaromatic amino acid biosynthesis in Bacillus subtilis. 1. Purifi-cation and properties of DAHP synthase. J. Biol. Chem.241:3365-3372.

11. Jones, S. T., R. A. Korus, W. Admassu, and R. C. Heimsch.1984. Ethanol fermentation in a continuous tower fermentor.Biotechnol. Bioeng. 26:742-747.

12. Kane, J. F., and M. J. Fiske. 1985. Regulation of phenylalanineammonia-lyase in Rhodotorula glutinis. J. Bacteriol.161:963-966.

13. Kimball, R. F. 1978. The reaction of repair phenomena tomutation induction in bacteria. Mutat. Res. 55:85-120.

14. Lemmel, S. A., R. C. Heimsch, and L. L. Edwards. 1979.Optimizing the continuous production of Candida utilis andSaccharomycopsis fibuligera on potato processing wastewater.Appl. Environ. Microbiol. 37:227-233.

15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

16. Morris, D. L. 1948. Calorimetric determination of total carbo-hydrates. Science 107:254.

17. Nakatsukasu, N. M., and E. N. Nester. 1972. Regulation ofaromatic amino acids in Bacillus subtilis 168. J. Biol. Chem.247:5972-5979.

18. Ott, L. 1984. An introduction to statistical methods and dataanalysis, 2nd ed. PWS Publishers, Boston.

19. Patel, N., D. L. Pierson, and R. A. Jensen. 1977. Dual enzymaticroutes to L-tyrosine and L-phenylalanine via pretyrosine inPseudomonas aeruginosa. J. Biol. Chem. 252:5839-5846.

20. Pierson, D. L., and R. A. Jensen. 1974. Metabolic interlock:control of an interconvertible prephenate dehydratase by hydro-phobic amino acids in Bacillus subtilis. J. Mol. Biol. 90:563-580.

21. Pittard, O., and F. Gibson. 1970. The regulation of biosynthesisof aromatic amino acids and vitamins. Curr. Top. Cell. Regul.2:29-63.

22. Schoner, R., and K. M. Herrmann. 1976. 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase: purification, properties,and kinetics of the tyrosine-sensitive isozyme from Escherichiacoli. J. Biol. Chem. 251:5440-5447.

23. Shiio, I., K. Ishii, and K. Yokozeki. 1973. Production of L-tryptophan by 5-fluorotryptophan resistant mutants of Bacillussubtilis. Agric. Biol. Chem. 37:1991-2000.

24. Shiio, I., S. Sugimoto, and K. Kawarmura. 1982. Production ofL-tryptophan by azaserine resistant mutants of Brevibacteriumflavum. Agric. Biol. Chem. 46:1849-1854.

25. Shiio, I., S. Sugimoto, and M. Miyajima. 1974. Regulation of3-deoxy-D-arabino-heptulosonate-7-phosphate synthase inBrevibacterium flavum. J. Biochem. 75:987-997.

26. Shiio, I., S. Sugimoto, and M. Nakagawa. 1975. Production ofL-tryptophan by mutants of Brevibacterium flavum resistant toboth tryptophan and phenylalanine analogues. Agric. Biol.Chem. 38:627-635.

27. Smith, K. C. 1978. Multiple pathways of DNA repair in bacteriaand their roles in mutagenesis. Photochem. Photobiol.28:121-129.

28. Srinivason, P. R., and D. B. Sprinson. 1959. 2-Keto-3-deoxy-D-arabino-heptulosonic-7-phosphate synthase. J. Biol. Chem.243:716-722.

29. Sugimoto, S., M. Nakagawa, T. Tsuchida, and I. Shiio. 1973.Regulation of aromatic amino acid biosynthesis and productionof tyrosine and phenylalanine in Brevibacterium flavum. Agric.Biol. Chem. 37:2327-2336.

30. Sutherland, J. B., D. L. Crawford, and A. L. Pometto III. 1981.Catabolism of substituted benzoic acids by Streptomyces spe-

642 SHETTY ET AL.

on April 13, 2020 by guest

http://aem.asm

.org/D

ownloaded from

L-PHE PRODUCTION FROM STARCH BY B. POLYMYXA MUTANTS 643

cies. Appl. Environ. Microbiol. 41:442-448.31. Suzuki, M., A. Bergland, A. Unden, and C. G. Heden. 1977.

Aromatic amino acids production by analogue-resistant mutantsof Methylomonas methanolophila GR. J. Ferment. Technol.55:466-475.

32. Walker, G. C. 1984. Mutagenesis and inducible responses to

deoxyribonucleic acid damage in Escherichia coli. Microbiol.Rev. 48:60-93.

33. Yamada, S., K. Nabe, N. Isuo, K. Nakamichi, and I. Chibata.1981. Production of L-phenylalanine from trans-cinnamic acidwith Rhodotorula glutinis containig phenylalanine ammonia-lyase activity. Appl. Environ. Microbiol. 42:773-778.

VOL. 52, 1986

on April 13, 2020 by guest

http://aem.asm

.org/D

ownloaded from