526
Anal. Chem. 1986, 58,526-532
50-99-7.
LITERATURE CITED
81OC
*
c >
b-
o 4
5c
w
z
b-
4 4
w
cc 1
4
1
1
1
12
2 0
28
--I
3 6
L 44 E
DAYS Figure 3. Long-term stability of the enzyme electrode.
Sy,x= 2.904, and a correlation coefficient of r = 0.95 were obtained. Registry No. EDTA, 60-00-4;oxalate oxidase, 9031-79-2;oxalic acid, 144-62-7; succinic acid, 110-15-6; ascorbic acid, 50-81-7; acetaminophen, 103-90-2;dihydroxyphenylacetic acid, 102-32-9; ccystine, 56-89-3;L-lycine, 56-87-1;L-lactic acid, 79-33-4;D-glUCOSe,
(1) Hodgkinson, A. "Oxalic Acid in Biology and Medicine"; Academic Press: New York, 1977. (2) Vogel, A. I. "Textbook of Quantitative Inorganic Analysis"; Longmans: London, 1982, pp 243, 320, 577. (3) Pakel, G.; Florio, F. A. "Kirk-Othmer Encyclopedia of Chemical Technology", 2nd ed.; Wiley: New York, 1967; Vol. 14, pp 356-373. (4) Peacock, M.; Heyburn, P. J.; Robertson, W. G. Br. J . Uroi. 1978, 50, 449-454. (5) Galosy, R.; Clarke, L.; Ward, D. I . ; Pak, Cyc J . Urol. (Baltimore) 1879, 723, 320-323. (6) Wyngaarden, J. B.; Elder, T. D. I n "The Metabolic Basis of Inherited Disease"; Stanbury, J. B., Fredrickson, D. S., Eds: McGraw-Hill: New York, 1960; pp 449. (7) Archer, H. E.; Dormer, A. E.; Scowen, E. F.; Watts, R . W. E . Lancet 1957, ii:320. (8) Hodgkinson, A. Clin. Chem. (Winston-Salem, N . C . ) 1970, 76, 547-557. (9) Zerwekh, J. E.; Drake, E.; Gregory, G.; Griffith. 0.; Hofmann, A. F.; Menon, M.; Pak, C. Y. C. Clin. Chem. (Winston-Salem, N . C . ) 1983, 29, 1977-1980. (10) Shimazono, M.; Hayaishi, 0. J . Biol. Chem. 1857, 227, 151. (11) Srivastava, S.K.; Krishnan, P. S. Biochem. J . 1982, 8 5 , 33-389 (12) Chiriboga, J . Arch Blochem. Blophys. 1986, 116, 516-523. (13) Guilbault, G. G. "Handbook of Enzymatic Methods of Analysis"; Marcel Dekker: New York, 1977; pp 497. (14) Klibanov, A. M. Anal. Biochem. 1979, 93, 1. (15) Kobos, R. K.; Ramsey, T . A. Anal. Chlm. Acta 1980, 721, 111-118. (16) Fonong, T.; Rechnitz, G. A. Anal. Chim. Acta 1984, 758, 357-362. (17) Mascini, M.; Guilbault, G. G. Anal. Chem. 1877, 49, 795-798. (18) White, W. C.; Guilbault, G. G. Anal. Chem. 1878, 5 0 , 1481-1486. (19) Chiriboga, J. Biochem. Blophys Res. Commun. 1983, 7 7 , 277-282.
.
RECEIVED for review July 29, 1985. Accepted October 8, 1985.
Direct Observation of Trypto phan Biosynthesis in Escherichia coli by Carbon-I3 Nuclear h, agnetic Resonance Spectroscopy Dennis J. Ashworth,* Chi 5.Chen, and Desmond Mascarenhas Western Research Center, Stauffer Chemical Company, 1200 South 47th Street, Richmond, California 94804
Carbon-I 3 nuclear magnetic resonance (NMR) spectroscopy has been applled to the dlrect monltorlng of L-tryptophan biosynthesis In genetically modlfled E . GO//. Growth of the followed by NMR bacteria In the presence of ~-[3-'~C]serlne analysis of the culture supernatant generated a spectrum contalnlng resonances from nonmetabollred [3-"C]serlne as well as resonances from serlne-derived [3-"C]tryptophan and [2-13C]acetate. Growth In the presence of [2-%]glycIne resulted In a spectrum contalning SIXmajor resonances. A comparison of the chemlcal shlfts to those of L-tryptophan allowed assignment of two resonances to [2-%]tryptophan and [3-13C]tryptophan. The remalnlng four resonances, generated by one-bond 13C-'3C coupllng ( J = 33.8 Hr), were asslgned to [2,3-13C]tryptophanand verlfied by two-dlmenslonal homonuclear ( ''C) correlated spectroscopy. Growth of the bacteria In the presence of [6-'3C]glucose resulted In the labeling of the C-3, C-4', and C-7A' posltlons of tryptophan. To monitor tryptophan productlon In a fermentor, a device was constructed that allowed the contlnuous pumping of ferment dlrectly Into and out of a speclal NMR tube whlle growth of the culture was maintained.
Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the more powerful methods for studying biological processes. This is due primarily to the technique's
nondestructive nature and the ability of a single NMR spectrum to reveal the many potential metabolites of a given substrate. Its ability to probe the biochemical pathways of plants (1-3), animals (4-6), yeast (7-9), and bacteria (10-12) is now well documented. In E. coli the immediate precursors of tryptophan, indole-3-glycerol phosphate and L-serine, have been well established from a number of investigations (13-15). The required serine is generally considered to be derived from 3phosphoglycerate. Following dehydrogenation, transamination, and finally phosphate hydrolysis of 3-phosphoserine, L-serine is obtained (16,17). An alternative route to serine is from glycine (18). Indole-3-glycerol phosphate is generated by the combination of erythrose 4-phosphate and phosphoenolpyruvate ultimately produce chorismic acid. Conversion of chorismate to anthranilate followed by addition of 5-phosphoribosyl l-pyrophosphate and indole ring formation then leads to indole-3-gycerolphosphate (19,ZO). Glucose, being the primary bacterial source of reduced carbon, is the origin of both serine and indole biosynthesis.
L-Tryptophan
0003-2700/88/0358-0526$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
Table I. Modified cogel-Bonner Medium (PH7.0) MgS04.7H,0 citric acid 80% H3P04 KCl NH4OH glucose N-Z amine AT'
0.2 g 2.0 g
5.4 g 4.0g 2.0 g 50.0 g 10.0 g
thiamineHC1 biotin FeSO4.7H2O MnS04 ampicillin anthranilic acidb distilled water
17.5 pg 3.5 Pg 1.0 mg 1.0 mg 30.0 mg 1000.0 mg 1.0 L
Purchased from Shefield Products, Memphis, T N . Neutralized with NH40H before use.
527
NMR tube a t 15 revolutions/s. The aeration device consisted of a 75 cm long, 3 mm wide hollow glass tube, which was inserted into the 10-mm NMR tube/culture and supported at the top of the magnet access port by a rubber stopper. Regulated air was then pumped into the spinning NMR tube via the glass tube. As a control for the aerated NMR tube culture experiment, an equivalent concentration of [2-13C]glycine (1000 ppm) was added to the remaining 9 mL of culture in the flask from which the cell aeration experiment sample had been obtained. This culture flask was then incubated under standard conditions (280 rpm, 30 "C) while the aerated NMR tube culture experiment proceeded. After 24 h, tryptophan production and viable cell counts were determined for both the NMR tube and shake flask cultures. [2-'3C]Glycine (90%) was purchased from MSD Isotopes and DL- [3-13C]serinewas synthesized essentially by the procedure of King (24) except for the substitution of [13C]paraformaldehyde (99%) in the synthetic scheme. Glucose Metabolism. Strain 2, which was similar to strain 1 but also required phenylalanine and tyrosine for growth, was grown in MVB (Table I) containing 1.5% glucose and 20 gg/mL each of tetracycline (TET), chloramphenicol (CAM), and ampicillin (AMP) and lacking anthranilate. For aerated growth of the bacteria in a 10-mm NMR tube, 0.1 mL of an innoculum preserved in 40% glycerol at -20 OC was added to 5 mL of MVB in a 25-mL flask. The culture was then grown overnight a t 37 "C to approximately 3 X lo9 cells/mL. From this culture 0.6 mL was taken and added to 5.4 mL of fresh MVB (less anthranilate), which was 1.0 mM in isopropylthiogalactoside. To this culture was then added 30 mg of [6-13C]glucosein 1 mL of DzO and the 7.0 mL was transferred to a 10-mm NMR tube. Following insertion of the NMR tube into the magnet, aeration a t 30 mL/min and spinning at 15 revolutions/s were commenced. Time averaged spectra were then acquired every 3 h for 45 h. [6-13C]G1~c~~e (90%) was purchased form Cambridge Isotope Labs. Continuous monitoring of 2-L fermentation cultures was performed by the addition of an exit and return port to the top of a New Brunswick Scientific Co. 2-L Bioflo fermenter jar with the exit tube submerged below the medium. Circulation of the culture out of the jar, into the modified NMR tube, and back into the jar was driven by a Masterflex peristaltic pump equipped with a 7014 head. The cycle time from jar exit to return was 1.5 min. NMR Spectroscopy. All carbon-13 NMR spectra were obtained on a Varian XL-200 spectrometer equipped with a broad-band probe operating at 50.1 MHz. The one-dimensional 32K supernatant spectra were obtained at 30 "C employing an 11-kHz spectral width (1.45-s acquisition time), 8.8-ps pulse width (50" pulse angle) with continuous broad-band 'H decoupling for 35K transients. The two-dimensional homonuclear (13C) correlated (COSY) spectrum was obtained from a 1.6-mL supernatant sample concentrated to dryness, resuspended in 0.5 mL of D,O, and placed in a 0.5-mL microcell. A a/2-tl-*/2 acquisition pulse sequence ( ~ / = 2 16.5 ps) was employed with appropriate phase cycling for quadrature detection in the second domain and continuous 'H decoupling. A 7000-Hz spectral width was used in both dimensions with the evolution period ( t l )incremented over 256 equidistant values to finally produce a zero-filled 1024 X 512 data matrix. All spectra were referenced to an external capillary solution of 8.9 M sodium formate (172.0 ppm) and resonance assignments were based on authentic sample chemical shifts and spiking the in vivo generated samples with authentic compounds.
l__-_l_
In the course of modifying E. coli for enhanced tryptophan production, the evaluation of t h e effect of each genetic modification was necessary. The increased tryptophan production by the bacteria provided a unique opportunity not only t o observe tryptophan production by carbon-13 NMR spectroscopy but, in addition, to observe the pathway through which the substrates flow to generate the find products. This has been attempted by the use of the specifically labeled substrates, [2-13C]glycine,[3-13C]serine,and [6-13C]glucoseand the application of two-dimensional (2-D) homonuclear (13C) correlated spectroscopy t o verify the carbon-13 labeling pattern. In addition, a cell aeration device is described that allowed growth of the bacteria in a 10-mm NMR tube so metabolism of [2-13C]glycine and [6-13C]glucose could be continuously monitored. Finally, a device is described that allowed continuous monitoring of glucose consumption and tryptophan production in a 2-L fermentor by carbon-13 NMR spectroscopy.
MATERIALS AND METHODS Glycine and Serine Metabolism. Strain 1,which was derived from E. coli K-12 and deficient in tryptophanase, was grown in a modified (Table I) Vogel and Bonner medium (MVB) (21). To initially establish the effect of serine addition on tryptophan production, an inoculum prepared in MVB was added to 20 mL of MVB medium at 0.13% in a 250-mL nephelo flask. The culture was then incubated at 30 "C (optimum for tryptophan production in this strain) and 280 rpm. When the density reached 500 Klett units (approximately 24 h), the culture was divided into two portions. One culture served as a control and the other received 1.0 g/L serine. After 24 h both culture's growth, tryptophan and indole production, and cell viability were compared. Tryptophan concentrations were determined by using the colorimetric method of Spies and Chambers (22). Indole was measured colorimetrically by the method of Smith and Yanofsky (23)and viable counts were determined using L-agar by a standard plate count procedure. Turbidimetric growth was expressed in Klett units by measuring the culture broth in a Klett and Summerson colorimetric using a green filter. For NMR spectroscopic evaluation of serine and glycine metabolism, 0.2 mL of a 24-h inoculum culutre (2.0 X lo9cells/mL) was added to 15 mL of MVB in a 250-mL Nephelo flask. After 24 h of growth, 1000 mg/L of either [2-'3C]glycine or DL-[313C]serinewas added to each culture flask. Depending on which precursor the flask received, culture samples for NMR analysis were carried out as follows: Serine. Two 1.6-mL culture aliquots were collected a t 0 and 24 h after serine addition. Each sample was then centrifuged a t 15000 rpm and 1.0 mL of supernatant collected. To this was added 0.5 mL of DzOprior to NMR analysis in a 5-mm tube. Glycine. In addition to the above experiment, an experiment was performed that allowed growth of a bacterial culture in an NMR tube while glycine metabolism was monitored. For this experiment, 6.0 mL of a 24-h culture (2.0 X lo9 cells/mL) was added directly to a sterile 10-mm NMR tube. Seven milligrams of [2-13C]glycinein 1.0 mL of D20 was then added to the NMR tube containing the bacterial culture. Following insertion of the NMR tube into the magnet, a culture aeration device was inserted into the medium. Time-averaged spectra were then acquired every 4 h during which time aeration and mixing were provided by introducing air to the culture a t 30 mL/min and spinning of the
RESULTS Two strains of modified E . coli K-12 were used in the experiments presented here. Strain 2, which required only glucose for high tryptophan production, was used to observe the incorporation of the carbon-13-labeled C-6 of glucose into both the amino acid side chain and the indole ring of tryptophan. Strain 1 required the addition of anthranilate to the medium for high indole production and therefore high tryptophan production. However, because of the rapid conversion of anthranilate t o indole in this strain, the addition of serine would also be expected t o enhance tryptophan production (recalling that serine and indole are both required for tryptophan production). As can be seen in Table 11, a significant improvement in tryptophan production was achieved upon
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
528
Table 11. Effect of Serine Addition on Tryptophan Production by Strain 1
treatment
growth, amt of TRP, amt of KU g/L indole, g/L
viable cells/mL
control
550
1.18
0.22
3.8 x 109
+l.OOg/L
540
2.18
0.11
4.4 x 109
A
~
SER
I
62 PPM
I
100 PPM
I
so
I
60
,
40
I
20
Flgure 1. I3C NMR spectra of E . coli culture supernatants (A) immediately following DL-[ 3-'%]serine addition and (B) 24 h after addition: SER, DL-[3-I3C]serine; TRP, L- [3-13C]tryptophan; AcO-, [2-'3C]acetate. Downfield resonances are due to a- and @glucose, and peak intensities in both spectra are relative to that of the 0-o-glucose C1
intensity (most downfield resonance). L-Tryptophan and acetate assignments are based on spiking the supernatant sample with authentic L-tryptophan and sodium acetate.
3k8
i0
26
Figure 3. Carbon-13 NMR spectrum of 1.0 mL of E. coli culture supernatant following 24 h of treatment with 1000 mg/L (15 mg) [2-13C]glycine;2, ~-[3-'%]tryptophan;5, ~-[2-'~C]tryptophan; 1, 3, 4, and 6, ~-[2,3-'~C]tryptophan; GLY-C,, [2-13C]glycine;GLU-C,, natural abundance C-6 of a- and P-o-glucose.
Table 111. Comparison of Tryptophan Production and Cell Viability in Shake Flask and Aerated NMR Tube Cultures of E . coli 24 h after [2-l8C]Glycine Addition"
shake flask NMR tube
amt of tryptophan, mg/L
viable cells/mL
3220
6.3 x 109 6.5 x 109
3940
"Viable cells/mL in culture flask and NMR tube at t = 0 h were
1.9 x 108.
Flgure 2. E . coli biosynthetic pathway for generation of observed carbon-13 labeled L-tryptophan from ~~-[3-'~C]serine and [2-I3C]glycine. Asterisk denotes carbon-13 label.
supplementation with serine. Notice also that higher tryptophan production was associated with lower indole accumulation (225 vs. 108 mg/L). Therefore, treatment of the bacteria with DL- [ 3-13C]serinewould be expected to provide a way of introducing a label into tryptophan so as to evaluate the effect various genetic modifications had on tryptophan production by 13C NMR spectroscopy. A comparison of the carbon-13 NMR spectra of the E. coli culture supernatant immediately following the addition of loo0 ppm DL-[3-'3C]serine to a 15-mL culture (2.1 x io9 cell/mL) and 24 h after addition (Figure 1) shows the conversion of L-serine (60.8 ppm) into L-tryptophan (27.0 ppm) with the C3 of L-serine becoming the C3 of L-tryptophan (Figure 2). The assignment of the 27.0 ppm resonance to L-tryptophan was based on spiking the supernatant sample with authentic tryptophan and the knowledge of the enhanced tryptophan producing ability of the organism. Also present in the spectra are natural abundance resonances from D-glucose (present as a carbon source in the medium) and one additional resonance in spectrum 1B corresponding to [2-13C]acetate (23.3 ppm). While it was not possible in this experiment to spectroscop-
ically resolve the DL-serine isomers present from the [313C]serinesynthesis, the conversion of D-serine to acetate via pyruvate has been reported by Dupourque et al. (25) and Labouw and Robinson (26). Treatment of the bacteria with 1000 ppm [2-13C]glycinefor 24 h followed by NMR analysis of the supernatant revealed a carbon-13 spectrum consisting of essentially six resonances derived from the Cz of glycine with little ( < l o % ) of the eubstrate remaining (Figure 3). Based on previously obtained chemical shifts of tryptophan, it was possible to assign resonance 2 (27.0 ppm) to ~ - [ 3 - ~ ~ C ] t r y p t o p and h a n resonance 5 (55.6 ppm) to ~-[2-~~C]tryptophan. The remaining four resonances (1,3,4, and 6), generated by one-bond 13C--13Ccoupling (J = 33.8 Hz), were assigned to ~-[2,3-~~C]tryptophan following 2-D homonuclear (13C) correlation spectroscopy to confirm the Cz-C3 coupling (Figure 4). The magnitude of the Cz-C3 coupling constant (33.8 Hz) was verified by performing a 13C-13C spin-spin coupled (inadequate) experiment on authentic L-tryptophan. The biosynthetic origins of these three labeled tyrptophan molecules are outlined in Figure 2. Because of the rapid conversion (>go% in 24 h) of the exogenous glycine to tryptophan observed in the initial shake flask experiments, it was felt that the course of the biosynthetic reaction might be readily observed by NMR spectroscopy. T o achieve this, a simple bacterial culture aeration device was constructed that allowed sustained cellular growth and metabolism in the NMR tube (see glycine experimental). A comparison of the NMR tube and shake flask cultures after 24 h (Table 111) shows that the NMR tube culture was maintained under adequate conditions for continued bacterial growth and metabolism. The resulting NMR spectra from the aerated NMR tube experiment are shown in Figure 5 . Observed are the increasing intensities of the labeled tryptophan resonances as well as the decrease in the [2-13C]glycine resonance as a function of time. While any nuclear Overhauser
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
529
GLY-C2 TRP-C2
TRP-C3
+ 0-4 HR.
A
mo
j I I
I
60
...-. ...-. ... .
Lm i
m
m
m
2000
15m
IWO
l
I
20 PPM
37 30
50 47
Flgure 5. E . coli metabolism of [2-13C]glycine. spectra were recorded every 4 h for 24 h while the NMR sample ( E . coli culture) was continuously aerated. Spectra are plotted in the absolute intensity mode. Tryptophan spectra are displayed at 10 times the vertical expansion of the glycine spectra. Resonance assignments are the same as in Figure 3.
1LN~
/
HZ
F1
Flgure 4. Two-dimensional homonuclear (13C) correlated (COSY) spectrum of an E . coli culture followipg treatment with 1000 mg/L [2-'3C]glycine for 24 h. The sample was obtained from 1.6 mL of supernatant concentrated to dryness, resuspended in 0.5 mL of D20, and placed in a 0.5-mL spherical microcell. Offdiagonal resonances verify the presence of ~-[2,3-'~C]tryptophan. Also present on the diagonal are a- and P-D-glucose, ~-[3-'~C]tryptophan, ~-[2-'~C]tryptophan, and residual [2-13C]glyclne.
effect enhancement of signals was not taken into account, a plot (not shown) of the total labeled tryptophan intensity and the [2-13C]glycinepeak intensity vs. time qualitatively supports the production of tryptophan a t the expense of intracellular glycine. Further characterization of the tryptophan biosynthetic pathway was obtained by growth of the bacteria in the presence of [6-13C]glucose. Figure 6 shows the 13C NMR spectrum of a typical aerated NMR tube bacterial cultrue immediately following the addition of 30 mg/7.0 mL [613C]glucose(Figure 6A) and 54 h later (Figure 6B). While a number of minor resonaqces in spectrum B are not assigned to specific compounds, clearly observed are three major resonances corresponding to carbqn-13 enrichment at the C-3 (27.1 ppm), (2-4' (118.9 ppm), and C-7A (136.8 ppm) positions of tryptophan. The specific biosynthetic reactions leading to 13Cenrichment at these three positions are suggested in Figure 7. T o determine the distribution of carbon-13 among the three labeled positons of tryptophan, a spectrum was obtained under conditions of no nuclear Overhauser enhancement of resonance intensities (gated decoupling) and appropriately long pulse delays (60 s) such that nonequivalent spin-lattice ( T I )relaxation of enriched carbons was of no consequence. This experiment generated C-3, C-4', and C-7A' resonances of equal intensity and therefore semiquantitatively supported the equal incorporation of 13Clabel into all three tryptophan positions.
DISCUSSION The high tryptophan producing strains of E. coli investi-
A
I
(SJ3C) Glucose
B (4"f) Trp
(3%) Trp
nknrcukI.( I
I
140
110
ao
I
50
20 PPm
Figure 6. E . coli metabolism of [6-13C]glucoseto [3-'3C]tryptophan, [4'-'3C]tryptophan, and [7A'-'3C]tryptophan. Bacteria were grown in an aerated NMR tube culture while time-averaged spectra were acquired every 3 h. Spectrum A was taken immediately following addition of 30 mg/7 mL [6-'3C]glucose. All resonances appearing in the spectrum can be assigned to natural abundance carbon signals from a- and @-glucosepresent in the medium and the two ( a and @) intense carbon-13 enriched C-6 resonances of glucose. Spectrum B was taken 54 h after the initial addition of glucose. Some minor resonances not assigned in spectrum B can be assigned to carbon-13 enrichment of phenylalanine. Spectra are plotted in the absolute intensity mode. The @- and a-[6-13C]glucose resonances in both spectra are displayed off-scale and are actually 28 times more intense than the two most downfield glucose resonances in spectrum A.
gated here provided a unique opportunity for observing tryptophan production by carbon-13 NMR spectroscopy.
530
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
HCOH HCOH
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'
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7i' YHZ HOFH HFOH HCOH %H,OP DAHP
4, TRYPTOPHAN ANTHRANILATE
HO 3-DEHYDOWINATE SHlKlMATE
Flgure 7. E . coli biosynthetic pathway for generation of [3-'3C]tryptophan, [4'-13C]tryptophan,and [7A'-13C]tryptophanfrom [6-'3C]glucose. The specific carbon that becomes the final labeled tryptophan carbon (3, 4', or 7A') is labeled in each intermediate. All intermediates are not shown and only one potential route for incorporation of label into the 7A' position of tryptophan is shown. GLY-3-P, glyceraldehyde 3-phosphate; DAHP, 3-deoxy-~-arabinoheptulosonicacid 7-phosphate; PEP, phosphoenolpyruvate; PRPP, 5-phosphoribosyl 1-pyrophosphate.
While the bacteria contained genetic modifications for enhanced tryptophan production, the normal biosynthetic pathway from glucose to tryptophan was unaltered. These experiments would have been particularly difficult in wild-type E. coli due to the low accumulation of the amino acid. The results obtained from growth of the bacteria in the presence of ~ ~ - [ 3 - ' ~ C ] s e rverified ine that tryptophan production could be observed by carbon-13 NMR spectroscopy. The exact origin of the [2-13C]acetate resonance observed consistently in the spectra is not certain. Whether the acetate is derived from D-serine or L-serine was not established due to the lack of pure D-[3-13C]Serineor ~-[3-'~C]serine. Both D-serine dehydratase (EC 4.2.1.14) and L-serine dehydratase (EC 4.2.1.13) are known to be present in E. coli (25, 26,31). The lack of a labeled acetate resonance in the supernatant spectra of the [2-13C]glycinetreated culture suggests that the compound may be derived primarily from D-serine, since the conversion of glycine to L-tryptophan must proceed via L-serine to generate the observed tryptophan labeling pattern and to provide a substrate for L-tryptophan synthetase (EC 4.2.1.20). However, the NMR spectrum following [2-13C]glycinetreatment also fails to show a serine resonance (Figure 3). This suggests that once the L-serine is formed from glycine, it may be immediately converted to L-tryptophan. This furthermore suggests that if the acetate resonance observed in the DL-3-13C]Serine treated culture was derived from L-serine, then either the binding constant (KM)of L-serine dehydratase for L-serine is
considerably lower than the binding constant of L-tryptophan synthetase for L-serine or the velocity of L-serine conversion to L-tryptophan is greater than the conversion of L-serine to acetate. The generation of the doubly labeled [ 2,3-13C]tryptophan observed here is particularly informative spectroscopically and biosynthetically. While the presence of adjacent carbon-13 atoms in the in vivo generated tryptophan was suggested by the pair of doublets in the one-dimensional spectrum (Figure 3), direct confirmation of the labeling pattern was definitively obtained by the 2-D COSY experiment (Figure 4). By observation of the off-diagonal resonances, the predicted pattern of correlation and therefore coupling between C2 and C3 of tryptophan was established. Biosynthetically, the presence of the [2,3-13C]tryptophansuggests a specific route in which two exogenous molecules of [2-13C]glycineenter the bacterium and combine to form one molecule of ~-[2,3-~~C]serine. The first glycine is decarboxylated and deaminated to generate which then labeled N5,N10-methylene(13C)tetrahydr~folate combines with a second labeled glycine molecule in the presence of L-serine: tetrahydrofolate 5,lO-hydroxymethyl(18). The transferase (EC 2.1.2.1) to generate ~-[2,3-~~C]serine labeled serine then combines with indole-3-glycerolphosphate in the presence of tryptophan synthetase to produce the L[2,3-13C]tryptophan product (Figure 2). While information regarding the events leading to the production of serine from glycine is certainly useful and allows
ANALYTICAL CHEMISTRY, VOL 58, NO. 3. MARCH 1 9 8 6
ti%] GLUCOSE 96.5 ppm
A
I
0-3
6
9
12
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21
24
27
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(e)
[4‘!”C] TRYPTOPHAN 118.9ppm
I
I 120 ll8ppm
Flgure 8. Direct observation of [4’-13C]tryptophanproduction and glucose consumption by modified E. coli. Time-averaged spectra were acquired every 3 h for 45 h while bacteria were grown in an aerated 10-mm NMR tube. Spectra expansions show (A) the decrease in the natural abundance C1 resonance intensity of @-c-glucosein the media as a function of time and (B)the incorporation of 13C label from [6‘3C]glucose into the C 4 ‘ position of tryptophan as a function of time. All resonances are displayed in the absolute intenslty mode and the glucose expansions are displayed at 1.5 times the vertical scale of the tryptophanexpansions. Initial concentrationof glucose in the medium was 350 mg of unlabeled and 30.0 mg of [6-13C]glucosein 7 mL. The final concentration of tryptophan in the NMR tube was 2.0 mg/mL. Resonance intensities are not corrected for any nuclear Overhauser effects enhancement of signals. a convenient method of 13C labeling newly synthesized tryptophan, little information is gained as to the effect various genetic modifications have on indole biosynthesis. Furthermore, when one supplements a culture with glycine or serine, the potential may exist for abnormally high tryptophan production from the artifically increased glycine or serine pools as observed in Table 11. The use of 13C labeled glucose, however, not only provides a substrate for both the serine and indole-3-glycerol phosphate biosynthetic pathways but also does not alter the normal media glucose concentration. An examination of the pathway (32) through which glucose is metabolized to generate tryptophan suggests that [6-13C]glucose should be the labeled substrate of choice as [1-13C]glucose almost immediately loses the C-1 labeled carbon as COz in the conversion of 6-phosphogluconate to ribulose 5phosphate. The results obtained here suggest that the biosynthetic pathway from [6-’3C]glucose to tryptophan follows the steps outlined in Figure 7 (33-35). The use of [6-’3C]glucosetherefore provided a method of monitoring the balance of serine production from glucose by the production of [3J3C]tryptophan and indole-3-glycerol phosphate production from glucose by the production of [4’-13C]tryptophan. Perhaps most interesting was the presence of enrichment in the C-7A’ position of tryptophan. While a number of events might lead to labeling in this position, perhaps the most reasonable pathways involve either the conversion of some 3-phosphoglycerate to phosphoenolpyruvate during serine biosynthesis or the recycling of the labeled glyceraldehyde 3-phosphate produced in the final step of tryptophan biosynthesis back into glycolysis where once again 3-phosphoglycerate could be converted to phosphoenolpyruvate. In either event the [3-13C]ph~sph~eno1pyr~~ate is then available to combine with erythrose 4-phosphate in the first step of aromatic amino acid biosynthesis to ultimately generate
531
[7A’-13C]tryptophan. The production of [4’-13C]tryptophan and the consumption of glucose as a function of time are shown in Figure 8. Brief mention should be made of a useful device that has allowed the continuous 13C NMR spectroscopic analysis of bacterial cultures grown in standard 2-1, fermentors (diagram available upon request). The utility of the device and of continuous NMR analysis lies in the fact that neither chemical. enzymatic, nor supernatant analysis of glucose or tryptophan concentration in the fermentor is necessary. By simply pumping ferment continuously out of the fermentor, into the NMR tube, the back into the fermentor, no interruption in culture growth or special assay procedure is necessary. Furthermore, because analysis can be performed continuously and the resulting spectra automatically stored, no decrease in glucose or other substrates being examined should go undetected and a complete record of the metabolic events of the particular fermentation under investigation is maintained. Finally, the production of specifically 13C enriched L-tryptophan obtained here may be of some utility in itself since this material could be used in structural studies of enzymes and as precursors in other metabolic studies.
CONCLUSION The data presented here represent an application of NMR spectroscopy to the analysis of metabolism in a genetically modified microorganism. The ability of NMR spectroscopy to allow the direct monitoring of the conversion of a primary carbon source to a specific end product demonstrates the power and usefulness of the analytical technique. In the conversion of [6-’3C]glucoseto tryptophan, the incorporation of label into the C-3, C-4’, and C-7A’ positions observed here by 13C NMR spectroscopy would have been impossible to detect using 14C-labeledglucose unless extensive degradation of the tryptophan was performed. Even the use of [2-I4C]glycine to follow tryptophan production would have led to erroneous conclusions due to the generation of the doubly labeled [2,3-13C]tryptophan observed here. The ability of the cell aeration device and continuous fermentation analysis device employed here to allow NMR spectral acquisition while cell viability is maintained demonstrates the possibility for monitoring other important anabolic and catabolic reactions in bacterial and potentially in plant and animal cell cultures. As observed elsewhere, cell oxygenation in combination with NMR spectroscopy can provide insight into biological processes not attainable from cell extraction or supernatant NMR analysis (27, 28). Furthermore, because NMR spectroscopy is a nondestructive technique, cells maintained in NMR tubes by oxygenation or profusion can be recovered for further growth or analysis if desired (29, ,301. The device used here allowed aeration of the bacterial culture medium in the fermentor while spectral acquisition proceeded. Therefore, spectral resolution was not diminished and the continuous flow of the bacterial culture into and out of the NMR tube prevented any significant settling of the bacterial cells. Future refinement in profusion device design may allow specific substrate or nutrient addition to cultures while spectral acquisition continues and potentially multiple spectroscopic analysis of rirculating plant or bacterial cell cultures into and out of the NMR tube while NMR analysis progresses. Work currently under way may allow resonance intensities to serve as “triggers” for automated substrate addition, such as glucose, to the culture broth or for increases or decreases in culture aeration or agitation.
ACRNO WLEDGMENT The authors are grateful to D .J. Brookman, J. Kirschbaum, C. K Tseng, and H. W Mvcrs at Stmffer for useful discussions
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Anal. Chem. 1986,58,532-535
Registry No. Tryptophan, 73-22-3; serine, 56-45-1; glycine, 56-40-6; glucose, 50-99-7.
LITERATURE CITED (1) Schaefer, J.; Skokut, T. A.; Stejskal, E. 0.; McKay, R. A.; Varner, J. E. J . Biol. Chem. 1981, 256, 11574-11579. (2) Martin, J.-B.; Bligny, R.; Rebeille, F.; Douce, R.; Leguay, J.-J.; Mathieu, Y.; Gum, J. Plant fhysiol. 1982, 70,1156-1161. (3) Ashworth. D. J.; Mettler, I.J. Biochemistry 1984, 23, 2252-2257. (4) Cohen, S. M.; Shuiman, R. G.; McLaughlin, A. C. f r o c . Nati. Acad. SCl. U . S . A . 1979,76,4808-4812. (5) Cohen, S.M.; Rognstad, R.; Shulman, R. G.; Katz, J. J . Biol. Chem. 1981, 256,3428-3432. (6) Balaban, R. S.;Gadian, D. G.; Radda, G. K.: Wong, G. G. Anal. Biochem. 1981, 116,450-455. (7) Dickinson, J. R.; Dawes, I. W.; Boys, A. S. F.; Baxter, R. L. Proc. Natl. Acad. Sci. U . S . A . 1983. 80,5647-5851. (8) Sillerud. L. 0.; Alger, J. R.; Shulman, R. G. J . Magn. Reson. 1981, 45. 142-150 (9) Den Hollander, J. A.; Behar, K. L.: Shulman, R. G. f r o c . Natl. Acad. Sci. U . S . A . 1981, 78,2693-2697. (IO) Ogino, T.;. Garner, C.; Markley, J. L.; Herrmann, K. M. froc. Natl. Acad. S o . U . S . A . 1982. 79.5828-5832. (11) Kerby, R.; Niemczura, W.;Zeikus, J. Gr J . Bacterid. 1983, 155, 1208-1 218. (12) Ugurbil, K.; Guernsey, D. L.; Brown, T. R.; Glynn, P.; Tobkes, N.; Edelman, 1. S. f r o c . Natl. Acad. Scl. U . S . A . 1981, 78,4893-4847. (13) Yanofsky, C.; Crawford, I. P. Enzymes (3rdEd.) 1972, 7 ,1-31. (14) Miles, E. W. Adv. Enzymol. Relat. Areas Mol. Biol. 1979, 4 9 , 127.- . i .a6 --. (15) Schnackerz, K. D.; Bartholmes, P. Biochem. Biophys . Res. Commun, 1983,111, 817-823.
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(16) Pizer, L. I.J . Biol. Chem. 1983,238, 3934-3944. (17) Umbarger. H. E.; Umbarger, M. A.; Siu, P. M. I. J . Bacteriol. 1983, 85. 1431-1439. (18) Stauffer, G. V.; Plamann, M. D.; Stauffer, L. T. Gene 1981, 14,63-72. (19) Tribe, D. E.; Pittar, J. Appl. Environ. Microbiol. 1979, 3 8 . 181-190. (20) Aiba, S.;Tsunekawa, H.; Imanaka, T. Appl. Environ. Microbiol. 1982, 43,289-297. (21) Vogel, H.;Bonner, D. J . Biol. Chem. 1956,278, 97-106. (22) Spies, J. R.; Chambers, D. C. Anal. Chem. 1947,20,30-39. (23) Smith, 0.; Yanofsky, 0. I n "Methods I n Enzymology"; Kaplan, N. O., Ed.; Academic Press: New York, 1962; Voi. 5, pp 794-806. (24) King, J. A. J . Am. Chem. SOC. 1947,69,2738-2741. (25) Dupourque, D.; Newton, A.; Snell, E. J . Biol. Chem. 1966, 241, 1233-1 238. (26) Labouw, R.; Robinson, W. J . Biol. Chem. 1966, 241. 1239-1243. (27) Navon, G.; Ogawa, S.;Shulman, R. G.; Yamane, T. Proc. Natl. Acad. Sci. U . S . A . 1977, 74,888-891. (28) Brown, F. F.; Jaroszkiewicz, G.; Jaroszkiewicz, M. J . Magn. Reson. 1983, 5 4 , 400-418. (29) Ugurbil, K.; Brown, T.R.; Den Hollander, J. A.; Giynn, P.; Shulman, R. G. froc. Natl. Acad. Sci. U . S . A . 1978, 75,3742-3746. (30) Foxall, D. L.;Cohen, J. S.J . Magn. Reson. 1983, 52. 346-349. (31) Crawford, I.P.; Ito, J. f r o c . Natl. Acad. Sci. U . S . A . 1981, 5 1 , 390-397. (32) Shii, I.; Ishii, K.; Yokozeki, K. Agric. Bid. Chem. 1973, 37. 1991-2000. (33) Crawford, I.P. Bacterid. Rev. 1975,39,67-120. (34) Berlyn, M. B.; Ahmed, S.I.; Giles, N. H. J . Bacteriol. Rev. 1970, 104, 768-774. (35) Gibson, F.; Pittard, J. Bacteriol. Rev. 1968,32,465-492.
RECEIVED for review July 30,1985. Accepted October 18,1985.
Measurement of Small Absorbances by Picosecond Pump-Probe Spectrometry G . J. Blanchard' Department of Chemistry, University of Wisconsin-Madison,
1101 University Avenue, Madison, Wisconsin 53706
M. J. Wirth* L-454, Lawrence Livermore National Laboratory, Livermore, California 94550
The absorptlon sensitlvlty of plcosecond pump-probe ground state recovery spectrometry is examined crltlcally. I t is found that the detection sensitivity Is limited by the quantum noise present on the probe laser. Thls uitrahlgh sensltlvlty Is attainable only with lasers producing transform limited pulses and a hlgh-frequency trlple modulation detection technlque. With a detection llmlt of 96 000 cresyl violet molecules In the sampled volume, lt Is shown that thls technlque Is comparable to other trace absorption methods for sensltlvlty and provldes the added selectivity of a two-color experiment.
Electronic spectroscopy is a well-established tool for a wide variety of analytical problems. One of its major assets is its potential for high sensitivity as well as small volume measurements. Fluorescence is recognized as being a more sensitive technique than absorbance. The major limitation of fluorescence measurements, however, lies in their range of applicability. Not all molecules of interest have an appreciable fluorescence quantum yield or exist in a nonquenching matrix. Thus, whereas all molecules absorb light at some wavelength, Present address: Bell Communications Research, Inc., 331 Newman Springs Rd., Red Bank, NJ 07701.
not all molecules emit light energy when excited. Absorption is therefore a more widely applicable but less sensitive technique than fluorescence. The sensitivity of conventional absorbance spectrometry is limited by the ability to measure small attenuations of a noisy source beam. The sensitivity limit of commercial instruments is approximately absorbance units. Efforts to improve absorbance sensitivity usually involve removing the contribution of the noisy source by converting absorbance into another type of signal, present on a less noisy background. Examples of this are thermal lens and photoacoustic measurements. The sensitivity limits of these techniques for liquid samples are typically on the lo4 absorbance scale ( I , 2). While these represent improvements over conventional absorbance spectrometry, such techniques do not measure directly the absorbance signal, making their applicability limited by the thermal expansion properties of the solvent. Direct measurement of absorbance could be made more sensitive if the noise on the source beam were reduced. Recent advances in dye laser technology have resulted in the achievement of a very low noise light source (3-5). The increased stability is accomplished by a process called mode locking. This process suppresses high-frequency fluctuations present on a free-running continuous wave (CW) laser by defining a fixed phase relationship between all of the active
0003-2700/86/0358-0532$01.50/00 1986 American Chemical Society
