Coordination of Glycolysis and TCA Cycle Reaction Networks

Coordination of Glycolysis and TCA Cycle Reaction Networks

Citrate-Glucose Cometabolism Eliminates Acids and Reveals Potential Metabolic Engineering Strategies"

A. GOEL! M. M. DOMACH," WILLIAM HANLEY,h J . W. LEE: AND M. M. ATAAIh,d hDepartment of Chemical Engineering

The University of Pittsburgh Pittsburgh, Pennsylvania 1521 9

"Department of Chemical Engineering Camegie Mellon University

Pittsburgh, PennJylvania 15213

INTRODUCTION

Significant benefits can accrue from improving the understanding of what mcta- bolic control strategies are utilized by microbes and how they are implemented. Investigating the regulatory features of the pathways that provide the raw materials and energy necessary for growth and product synthesis has been recognized as an important starting point. Consequently, numerous strategies havc been pursued that havc yielded useful information and insights.

The picture that has emerged in bacteria such as Escherichia coli and Bacrllus subtilis is that they tend to have mismatched glycolytic and oxidative capacities. Thc oxidative constraint may reside in the activity of a TCA cycle enzymc such as a-ketoglutarate dehydrogenase',2 or cytochrome chain-mediated electron transfer capacity.3 The consequence of the mismatch is that carbon utilization efficiency is diminished especially a t high growth rates, where glycolytic by-products arc excrcted to the medium. Additionally, excess ATP production and degradation may occur due to poor coupling between precursor formation by glycolysis and the TCA cycle.4

We have shown that the mismatch can be nearly eliminated in B. subtilis.f) Adding citrate to glucosc-minimal medium results in citrate-glucose comctabolism character- ized by reduced glucose uptake, nearly theoretical carbon yield (bascd on total glucose and citrate carbon), no acid production, and several-fold increase in recombinant protein productivity.' Moreover, thc decrcascd glycolytic flux is not associated with a lower growth ratc (the growth ratc, in fact, slightly increases). Finally, the oxygen requirement is drastically reduced; this outcome is significant to large-scale cultures.x Achieving high carbon yield and nil flux of glucose to acetate or other acids without growth ratc declining demonstrates that metabolic flux regulation can bc changed to improve efficiency. Restricted glucose flux was hypothesized to result, in part, from citrate addition directly or indirectly modulating phosphofructose kinase

'This work was partially supported by National Science Foundation Grants BCS-9222557

dTo whom correspondence should be addressed. and BCS-92077614 to M.M.A. and M.M.D.

1

2 ANNALS NEW YORK ACADEMY OF SCIENCES

(PFK) and pyruvate kinase (PYK) activities which have been observed in higher cells. Citrate is readily transported by B. subtilis9 and other gram-positive bacteria. The coupling between citrate transport and divalent metal ion uptake could alter the intracellular balance of metal ions. In particular, the elevation of Ca2+ concentration by citrate-Ca2+ cotransport would reduce pyruvate kinase activity because Ca2+ is a strong inhibitor of this enzyme both in prokaryotes and the eukaryotes.lnJ1 Lower PYK activities, in turn, increase the phosphoenolpyruvate (PEP) pool,12 which is a strong inhibitor of PFK in bacteria including E. coli and B. subtilis species.

Consistent with the corresponding flux values, the enzyme activity measurement presented in this paper reveals a general pattern of lower activities of enzymes of central pathways for the dual substrate feed (glucose plus citrate) than the single feed (glucose only) cultures. Of particular interest is the pyruvate kinase<atalyzed flux, which is drastically lower for the dual-feed experiments. In addition to present- ing the measured fluxes for various cultures, this paper also identifies the maximal properties of B. subtilis networks comprising the tricarboxylic acid (TCA), hexose monophosphate (HMP), and glycolytic reactions. The problem entails, in part, formulating metabolite balances and specifying biosynthetic loads based on cellular composition, which has been a fruitful strategy for analyzing other metabolic problems.2q13-i6 However, as opposed to assuming that capacity constraints are present (e.g., limiting enzyme flux, maximal respiration rate), flux assignments are identified that maximize an objective subject to the load and balance rules.i7 Maximizing substrate carbon conversion to cell mass (Yc), or minimizing PFK flux, are the objectives examined. The use of an objective function approach is akin to that used to identify different routings of glucose to fat in adipose tissue.lx These constrained, optimal solutions provide a sense of the extent to which the Y, exhibited during citrate-glucose cometabolism is suboptimal. Additionally, they allow for the identification of alternate flux routings capable of providing the Y,-observed. Addition- ally, these analyses reveal that HMP pathway cycling may occur via the reversible reaction catalyzed by glucose-6-phosphate isomerase and show that our experimental finding corresponds closely to the maximum potential conversion of substrate to cell mass. Finally, we present our results demonstrating that the dual feeding strategy leads to a significant increase in a recombinant culture productivity.

EXPERIMENTAL PROTOCOL

Bacterial Strains and the Medium

Wild-type B. subtilis (Marburg strain) was obtained from American Type Culture Collection (ATCC number 6051). This strain was a tryptophan auxotroph. One liter of medium contained: 1 g NH4CI; 0.04 g tryptophan; 1.0 g KHzPO4; 2.72 g K2HP04; 0.284 g Na2S04; 0.17 g NaN03; 0.15 g KCI; 25 mg MgC12 . 6H20; 2.16 mg FeCI3 . 6H20; 15 mg MnClz. 4H20; 22 mg CaCI2. 6Hz0; and 2.5 ml of 10% antifoam B. The concentrations of glucose and citrate used in each experiment are noted in the text. The B. subtilis strain BR151 (trp, met, lys), which contains a chromosomal copy of lad gene and carries plasmid p602/19 was used for recombinant cultures.iYJO The plasmid contains the cat (chloramphenicol acetyltransferase) gene downstream of a strong vegetative T5 promoter. The plasmid also provides resistance to kanamycin (pUB110-derived). The strain carrying the plasmid was kindly provided to us by Dr. LeGrice at Hoffman-La Roche & Co. Ltd., Basel, Switzerland. The medium for these studies was similar to that of the wild type with the following additions: 0.04g/l of methionine and lysine and 0.01 g/l of kanamycin.

GOEL et al.: GLYCOLYSIS AND TCA CYCLE 3

Reactor Setup

Fermentation vessels with 2-liter capacity (Applikon, Austin, TX) were used in this study. An on-line data acquisition system2' collected data for pH, agitation, air flow rate, and C 0 2 evolution rate. The air flow rate was set at 2 I/min, and pH was constrained between 6.7 and 6.9. The temperature was maintained at 37°C by using a water circulator (Cole Parmer, Chicago, IL). lnocula were grown in Luria-Bertani (LB) broth; the inoculum size used was 1% of reactor volume.

Cell Mass, Residual Glucose, and Extracellular Organic Acids Concentrations

These were measured exactly as described in Goel et a1.22

Intracellular Enzyme Assay

For analysis of intracellular enzymes, steady-state cell samples were taken by pumping a fixed volume (typically 100 ml) rapidly (within 10 sec) into a conical flask and chilling the sample using liquid nitrogen. When the temperature fell below 4"C, the sample was centrifuged for 10 min at 10000 rpm in a Sorvall RC2B superspeed centrifuge using a Sorvall SS-34 rotor a t 4°C to isolate the cell pellet. The resulting pellet was washed with 0.05 M Tris (pH 7.5) containing 100 mM KCI and stored at -70°C. Thawed cells (typically coming from 30 ml original culture) were resus- pended in the same buffer, and incubated at 37°C with 1 mg/ml lysozyme to lyse the cells (usually 15 min). All samples were aliquoted and stored at -70°C. Tris-HCI (50 mM, pH 7.5) was used to resuspend the pellets when needed. This procedure was similar to that used by Fisher and Magasanik.??

The enzymatic assays were performed in a Lambda 6 UV/vis spectrophotometer (Perkin Elmer, Nonvalk, CT). The change in absorbance of a species involved in the reaction catalyzed by the enzyme, directly or indirectly, is correlated to the level of that enzyme. Pyruvate carboxylase (PC) was measured according to the method described by Young et ~ 2 1 . ~ ~ Malate dehydrogenase (MDH) was measured using the method outlined by Y o ~ h i d a . ~ ~ Phosphoglucose isomerase (PGI) was measured using the method of Noltman.2h Pyruvate dehydrogenase complex (PDC) was measured by estimating the levels of one of its components, dihydrolopoyl dehydrogenase, using the method of Reed and W i l l m ~ . ~ ~ Malic enzyme was measured according to the method described by Hsu and Ldrdy.28 Glucose 6-phosphate dehydrogenase (GDH) was measured using Sigma diagnostics kit number 345-UV. CAT was assayed following the procedure of Rodriguez and Tait.2y

FLUX MODEL AND OPTIMIZATION ANALYSIS

Flux Values Using Experimentally Measured Parameters

The network typically assumed to encompass the central metabolic reactions used by B. subtilis is shown in FIGURE 1. The flow of carbon in this network is quantified by 31 flux quantities (r,, mmole/g-cell/hr). For this network, 15 balance equations for 31 fluxes can be generated:

r, = r2 + r3 + rl0 ( la)


Glucose

r1 i cell envelope

‘1 2 k

- ‘2 Ribulose 5P

YIUIUOG ilr / \ 4 r6 nucleicacid 1 3 5 1 3 4 aminn acid

113 lipid - TP

step which produces ATP

step which produces NADPH

amino acid rrS nucleic acid 3PG

I step which produces NADH

A step which produces FADH

lipid

47 aminoacid cell enveloDe

rLp

amino acid nucleic acid

‘3 0 4--

organic acid

amino acid, peptidoglycan

rZ2 lipid synthesis

CIT

amino acid 0 KG --+ polyamines

MAL

r27

y 53

succinate - Succ 4

FIGURE 1. Schematic of the pathways used in the flux calculations. (Malic enzyme-catalyzed raction is not included.) Acids other than acetate, including pyruvate, are represented by rlq.

rIu = rIl -k rI2 - r7 - r,

2r12 = rl.l r14 - r8

r14 = rl.5 -k r16

r16 = rl r17 r I X

r18 = r19 -k r20 -k r21 - rl -k r31

GOEL el af.: GLYCOLYSIS AND TCA CYCLE 5

'21 = r22 + r23 + '24

r24 = r25

'25 = r26 + r27

r27 = r28 + r29

r2 = r, + r,

r4 = r, + r7

r, = r7 + r,

r , = r, + r4

(1m)

(In)

(10) The effect of adding the NADPH requirement was determined by adding a new constraint to the model:

2r2 + r25 = 18 (1P) In Equation (Ip), the NADPH requirement is based on other's estimates for bacteria.3"

Eleven of the fluxes (r3, r6, r4, r l l , rl.3, ~ 1 5 , r17, r20, r22, ~ 2 ~ . and r.30) can be assigned to metabolic loads based on a typical cellular composition and known biosynthetic stoichiometries as has been done by Goel et ~ 1 . ~ ~

The values of four fluxes, rl, r19, r23, and 1 - 2 ~ are measured experimentally. Moreover, an equation for C02 evolution rate based on the above fluxes can be written. This leads to a total of 32 equations and 31 fluxes, which are solved using a least squares procedure. Other parameters that are calculated from the fluxes are:

ATPproduced = -rI2 + 3r14 + rlx + r2, + 3r2, + 2rz, + 3rZ9 - r.31 (2a)

= IOOODIATPproduced (2b)

Y, = S00D/72r1 (2c) In Equation 2a, it is assumed that there is a maximum of two phosphorylation sites. In practice, we assume that NADH and FADH yield 2 and 1 mole of ATP, respectively, when oxidized. In Eq. 2e, Y, (mg carbon in celllmg carbon consumed) was calculated by assuming that 50% of the cell dry weight is carbon. This equation is modified to include the consumption of citrate carbon in dual-feed cultures as given in later part of the paper (Eq. 5).

Optimization Studies

Equations l a through Ip and the biosynthetic loads result in 27 equations; hence, there are four degrees of freedom. Based on this formulation, the optimization problem becomes finding four flux assignments that maximize a yield subject to satisfying the balance equations. When only glucose is metabolized, the problem of maximizing Y, can, for example, be formulated as

MAX [Y'] = >MIN [r,] (3a)

(3b) s.t. A . r = b


s.t. r, 2 0 (3c)

s.t. r, = c (34 where A, r, and b denote the flux balance coefficient matrix, flux vector, and a constant vector. Additional restrictions can be added such as r; 2 0 (Eq. 3c), which requires that, for example, a reaction proceeds in its thermodynamically favorable direction. Alternately, a flux can be required to be a constant (i.e., no acetate overflow allowed) as Equation (3d) represents.

The solutions to the optimization problem were obtained by using Lindo soft- ware. In general, the problems fall in the class of linear formulations with linear constraints and objective functions that can readily be solved with a variety of linear programming methods.

RESULTS

Summary of Chemostat Experiments

The data for chemostat cultures fed only glucose is summarized in TABLE 1. In the 1 g/l glucose feed cases with no citrate (runs 1-3), acid formation was negligible,

TABLE 1. Yields and Acid Production of Cultures Fed with Glucose Expt. D SI, S2" Sir s21 C Acet Pyr a-KG Percent Percent No. (hr -9 ( d l ) ( d l ) (g/l) (g/l) Yield O.D. (g/l) (g/l) (g/l) Acid CO?

1 0.15 Gluc(1) - 0 - 0.29 0.7 0.0 0.0 0.0 0.0 60 2 0.3 Gluc(1) - 0 - 0.32 0.75 0.0 0.0 0.0 0.0 56 3 0.5 Gluc (1) - 0 - 0.35 0.80 0.0 0.0 0.0 0.0 56

5 0.4 Gluc (2) - 0 - 0.31 1.5 0.1 0.7 0 41 26 6 0.2 Gluc(4) - 1.4 - 0.19 1.2 0.2 1.52 0 67 16 7 0.4 Gluc (4) - Washout - - ~ - - -

NOTE: OD and D refer to the optical density measured at 660 nm and dilution rate, respectively. Gluc refers to feed glucose concentration (g/l). S,,,, S*,, refer to feed concentration of glucose ( 1 ) and added component (2). respectively. SIf, Szr refer to residual concentration of glucose and added component (2). respectively. C-Yield is defined as g carbon in cell/g carbon consumed from feed. % Acid is defined as g carbon as acids/g carbon consumed from feed. % CO, is defined as g carbon as C 0 2 / g carbon consumed from feed.

4 0.2 GIUC (2) - 0 - 0.30 1.5 0.2 0.1 00.0 15 49

- -

and the glucose carbon was converted to C 0 2 (60%) and biomass (40%). However, increasing the feed glucose concentration to 2 g/l resulted in the production of a significant amount of acid (runs 4-5). Increasing glucose concentration further to 4 g/l (runs 6-7) resulted in the incomplete utilization of glucose even at a low dilution rate of 0.2 hr-*; almost 70% of the metabolized glucose was converted to acids. Moreover, at a higher dilution rate of D = 0.4 hr-', the culture was unstable and washed out, probably as a result of excessive acid formation at the onset of the continuous run. The cell yield based on the carbon consumed is approximately 30% for 2 g/l glucose in the feed at both dilution rates of 0.2 hr-I and 0.4 hr-' and decreases to 19% for cells fed 4 g/l glucose at dilution rate of 0.2 hr-I. In summary, the results reveal that concentrations of acids increase significantly with both an increase in dilution rate and the feed glucose concentration, and culture cannot be stably maintained when glucose concentration is 4 g/l.

GOEL ef al.: GLYCOLYSIS AND TCA CYCLE 7

TABLE 2. Yields and Acid Production of Cultures Fed Glucose Plus Citrate Expt. D S,, S?a Sl i SZi c Acet Pyr a-KG Percent Percent No. (hr-I) (gil) (gil) (gil) (g/l) Yield" O.D. (gil) (g/l) (gil) Acid COz

8 0.2 Gluc(2) Citb (0.64) 0 0 0.57 3.7 0 0 0 0 44 9 0.4 Gluc (2) Cit (0.71) 0 0.1 0.63 4.0 0 0 0 0 40

11 0.2 Gluc (4) Cit (l.2Y) 0 0.25 0.61 7.5 0 0 0 0 43 12 0.4 Gluc (4) Cit (0.64) 0 0 0.59 6.5 0 0 0 0 40

10 0.4 Gluc (2) Cit (0.32) 0 0 0.73 4.1 0 0 0 0 27

"C Yield is defined as g carbon in cellig carbon in glucose and citrate. hCit refers to the addition of citrate to the glucose medium. Other headings are defined in TABLE I

To illustrate the effect of citrate addition, two experiments at dilution rates of 0.2 and 0.4 hr-l were conducted with feed concentrations of 2 g/l glucose and 0.64 g/l citrate (runs 8 and 9, TABLE 2). No acids were detected, and the cell yield was substantially enhanced. The experiment was also repeated with a lower citrate concentration in the feed (run 10). Again, no acids were detected in the culture supernatant, and a high cell yield was attained.

To determine if the instability that accompanies increasing feed glucose concentration was reduced, citrate-glucose co-feeding experiments with 4 g/l glucose in the feed were conducted. The results of these experiments (runs 11 and 12) revealed that no extracellular organic acids were detected. Rather, the glucose was completely utilized, and citrate was almost fully consumed for the two dilution rates examined (0.2 and 0.4 hr-I). These results contrast sharply with those found with a single glucose feed (see FIGS. 2 and 3) where washout occurred at D = 0.4 hr-' (run 7), and at D = 0.2 hr-I (run 6), about 70% of the metabolized glucose was converted to acid by-products.

To establish whether the lowered glycolytic flux results from citrate inhibition or citrate replacing the glucose used to form TCA cycle amphibolites, citrate was replaced with glutamine. The results (TABLE 3, run 13) showed that acid formation

1 ~ 1 ~ 1 ' 1 ~ 1 , 1 ' 1

8 - -

-

Dual Feed

6 -

4 - -

-

~ , , , , , I , I , , , , . ,

0 10 20 30 40 50 60 7 0 80

Time (h)

FIGURE 2. Optical density profiles for chemostat cultures fed with single feed ([glucose] = 4 g/l) and dual feed ([glucose] = 4 g/ l and [citrate] = 1.29 g/l) at a dilution rate of 0.2 hr-'.


0 Single Feed rn Dual Feed

Time(h)

FIGURE 3. Optical density profiles for chemostat cultures fed with single feed ([glucose] = 4 g/l) and dual feed ([glucose] = 4 g/l and [citrate] = 1.29 g/l) at a dilution rate of 0.4 hr-I.

was significant for the dual feeding of glucose and glutamine even at a glucose concentration of 2 g/l. Furthermore, experiments in which the glucose feed was supplemented with a complex mixture of nutrient broth and amino acids also exhibited acid formation (runs 14-15).

Intracellular Enzymes and Fluxes

The flux values (see TABLE 4) are lower for the dual-feed than the single-feed cultures. A flux directly relevant to our hypothesis is the flux of the pyruvate kinase-catalyzed reaction (rlx). This flux is drastically lower for the dual-feed experiment than the single feed. Moreover, the TCA cycle flux of citrate to a-ketoglutarate is also substantially reduced for the dual-feed experiments. Regarding the TCA cycle fluxes, it is interesting to note that the flux values for dilution rates of 0.2 hr-' and 0.4 hr-I are approximately equal for cells grown in thc presence of glucose, indicating a possible saturation of TCA cycle flux.'

Intracellular enzyme assays were performed for some selected enzymes (see TABLE 5). As a general pattern, the cnzymatic levels for the singlc feed were higher

TABLE 3. Yields and Acid Production of Cultures Fed Glucose Plus Glutamine and Other Nutrients Expt. D S , , s20 SII S2I c Acet F'yr a-KG Percent Percent No. (hr-I) (g/l) (gil) (g/l) (g/l) Yield O.D. (gil) (gil) (g/l) Acid C 0 2 13 0.4 Gluc(2) Gln(0.81) 0 0 0.35 2.3 0.5 0.24 0.4 28 30

- 14 0.25 Gluc(2) # 0 - 4.0 0.3 o n 15 15 0.5 Gluc(2) # 0 - 2.6 0.4 0 0 58" -

NOTE: Gln, and # refer to the addition of glutamine and nutrient broth, respectively, to glucose medium. "Other organic acids were lactate '0.3 g/l), Cormate (0.39 g/l), and acetoin (0.16 gil). Other headings are

defined in TABLE 1.


TABLE 4. Measured Fluxes (mmol/g cell-hr) for Single- and Dual-Feed Chemostats

Parameter Glucose uptake rl Glycolysis rl? Pyruvate kinase r / ~ Citrate synthase r24

Citrate uptake rc,, a-Ketoglutarate dehydrogenase r27 Pyruvate carboxylase r j l Total acid production rry + r23 + r28

Single Feed Single Feed Dual Feed Dual Feed D = 0.2 hr-l D = 0.4 hr-I D = 0.2 hr-l D = 0.4 hr-'

4.63 8.95 1.84 3.33 8.05 15.99 2.61 4.44 2.96 6.12 0.30 0.19 3.92 3.65 0.86 0.88

- 0.86 0.88 3.69 3.17 1.18 1.40 0.62 1.27 0.08 0.27 1.88 7.82 0.03 0.04

-

than for the dual-feed cultures. These lower enzyme activities for the dual-feed experiment are consistent with the lower flux values given in TABLE 4.

The malic enzyme proved to be different from the indicated pattern: its activity was significantly higher in the dual-feed scenario. As malic enzyme provides a pathway for consumption of citrate, adding citrate may derepress this enzyme, thereby providing a channel for consumption of the uptaken citrate. In our theoretical analysis, we have also investigated the pathway schematic that includes malic enzyme activity.

Theoretically Optimized Fluxes

As noted earlier, the optimization problem becomes finding four flux assignments that maximize a yield subject to satisfying the balance equations. Overall, these calculations (see TABLE 6, row a ) indicate that if regulatory and dissipative processes are insignificant (Le., zero maintenance energy), the network's output simultaneously fulfills cellular carbon and biosynthctic ATP requirements. That is, the network is intrinsically capable of providing the minimal carbon skeleton and ATP requirements; increasing glucose flux would only increase ATP and acid

TABLE 5. Intracellular Enzyme Specific Activity (nmol . mgP-' . min-1) for Single and Dual Feed

Enzyme Glucose 6-P dehydrogenase

Isocitrate dehydrogenase

Pyruvate carboxylase

Pyruvate dehydrogenase complex

Phosphoglucose isomerase

Malic enzyme

Feed 2 g/l Glucose D = 0.4 hr r ' 8290 f 13% (n = 3)o

20599 f 15%

13565 2 25% (n = 3)

18569 f 7% (n = 3)

72347 f 12% (n = 3) 2.2 t 12% (n = 3)

(n = 3)

Feed = 2 g/l Glucose and 0.64 g/l Citrate;

D = 0.4 hr- '

(n = 3) 5267 ? 8%

(n = 3) 2562 ? 6% (n = 2)

548 * 5% (n = 3)

28135 2 0 7 ~ (n = 3)

215 f 13% (n = 3)

8290 f 13%

"n represents the number of measurements.


TABLE 6. Fluxes and Yields for Network Alternatives with Positive Fluxes for D = 0.2 hr-I

Glucose Citrate Malic TCA Flux Flux Enzyme Flux ( r l ) (mil) Flux (~27)

(a) 1.73 NA NA 0 (b) 1.47 0.49 NA 0.53 (c) 1.59 0.27 NA 0.28 (d) 1.40 0.47 0.40 0.40 (e) 1.54 0.26 0.19 0.19

PYK Flux ( T I 8 1

0.20 0.02 0.10 0.00 0.07

~

Aceiate Flux ATP Carbon (rzj) Prod. Yield YAP 0.00 36.8 0.80 27.17 0.00 48.9 0.71 20.5 0.00 43.3 0.75 23.09 0.00 36.6 0.74 27.32 0.00 35.5 0.77 28.1

NOTE: Units for flux and carbon yield are mmol/g cell and g cell C/(g glucose C + g citrate C), respectively. To compute the carbon yield, cells were assumed to be 50% by weight carbon. TCA cycle flux refers to a-ketoglutarate dehydrogenase flux. ATP produced is mmol ATP/g cell. The optimal distribution of fluxes is independent of the dilution rate; however, fluxes are scaled according to the dilution rate.

production. Thus, the success of citrate-glucose cometabolism cannot be attributed to alleviating topological constraints. Rather citrate-glucose metabolism occurs by an alternate flux assignment that has high carbon yield. Moreover, citrate addition induced regulatory changes that permit the alternate flux assignment to be realized. Thus, further examination of citrate- and glucose-derived fluxes that satisfy MAX [Yc] 2 Y,-observed would suggest how carbon fluxes were rerouted. The results, discussed next, reveal that several versions of the central pathways can account for the high Y,-observed values. Regardless of the version, however, Y,-observed is comparable to MAX [Y,], indicating that the degree of suboptimality is low.

To establish the most efficient way to cometabolize glucose and citrate, a similar analysis was performed on the network shown in FIGURE 1, with an additonal external citrate flux r,,,, to the citrate pool. This was used to account for the utilization of exogenous citrate. The model representation remained similar to the single-feed scenario with the only change being in the citrate balance; Equation (li) was replaced by

‘cil + r24 = r25

where rclr denotes the rate of citrate transport. Because additional carbon in the form of citrate is present, Equation 2c is also modified to

(4)

Y, = 500D/72(r1 + rrjl)

The flux assignment shown in TABLE 6, row b, for a glucose/citrate ratio of 3 allows for a yield of 0.71 g cell C/g total substrate C, which exceeds the value observed of 0.63 g cell C/g total substrate C (run 9, TABLE 2). Fixing the ratio at 6 generates a MAX [Yc] = 0.75 g cell C/g total substrate C (see TABLE 6, row c). This is only slightly greater than the observed value of 0.73 g cell C/g total substrate C (run 10, TABLE 2).

These similarities between the MAX [Y,] and Y,-observed values suggest that the network assumed and the model parameters chosen can potentially account for the high carbon yield observed, and the degree of suboptimality in Y,-observed is small. However, these conclusions should be scrutinized further in light of the potential pitfalls of pathway analysis. Namely, alternate networks and flux distributions may exist that are feasible. Additionally, uncertainties in model parameter values will result in a confidence interval being associated with a MAX [Yc J value. The MAX [Y,.]


values for the alternatives may be greater, thus adding to their feasibility. Therefore, the pathway incorporating malic enzyme was also investigated (FIG. 4). For the same glucose/citrate ratio, including malic cnzyme activity increases MAX [Yc] (TABLE 6, rows d and e). To complete the investigation of alternate flux distributions, the effect of relaxing the directionality constraint for rill was investigated.

To this point, all reactions were constrained to operate in the directions shown in FIGURE 1 by requiring r, 2 0. Although this constraint is consistent with thermodynamics of many reactions or the biosynthetic load nature of the flux, the reaction rlo is

Glucose

rg I G6P - Ribulose 5P

‘1 2

113 lipid C-- TP

19 --t amino acid

step which produces ATP nucleic acid t 3PG amino acid r15

I step which produces NADH 0 step which produces NADPH A step which produces FADH

organic acid

amino acid, peptidoglycan

lipid

amino acid -PEP c7

cell envelope

- amino acid polyamines

MAL

‘2 7

.a KG succinate Succ 4

FIGURE 4. Same as FIGURE 1 but including malic enzyme.


TABLE 7. Fluxes and Yields for Network with Malic Enzyme and Reversible G6P Isomerase at D = 0.2 hr-I

G6P Glucose Citrate Malic TCA PYK Isom PFK

Flux Flux Enzyme Flux Flux Flux Flux ATP Carbon ( r l ) (rc,,) Flux (r27) (r18) (r10) (r12) Produced Yield

(a) 1.51 0.25 0.08 0.08 0.00 -0.14 0.78 31 0.79 h) 1.40 0.47 0.40 0.40 0.00 0.07 0.78 37 0.74

NOTE: Abbreviations: G6P lsom and PFK refer to glucose-6-phosphate isomerase (r,") and phosphofructose kinase (r12). Other headings defined in TABLE 6.

a reversible isomerization. Constraining the glucose-to-citrate ratio to 6 results in MAX [Yc] to 0.79 g cell C / g total substrate C (TABLE 7, row a ) , and HMP cycling is required as indicated by the negative value of rl0. Constraining the glucose-to-citrate ratio to 3 further reduces MAX [Y,] to 0.74 g cell C/g total substrate C and no HMP cycling is required. This flux distribution is equivalent to that obtained earlier (TABLE 6, row d). Overall, when thermodynamics and the observed Y, values are considered, a flux distribution that has malic enzyme flux and reversible glucose-6- phosphate isomerase flux is feasible and has the highest Y,.

Apart from allowing for a reverse reaction, the flux distributions in TABLE 7 have another common feature. They correspond to minimizing PFK flux to its lowest possible value. That is, the MAX [Yc] and MZN [rlz] flux assignments correspond. Thus, the distributions can be regarded as the ones feasibly approached if citrate addition directly or indirectly depresses PFK activity. Additionally, all the optimized flux distributions in the presence or absence of citrate yield a pyruvate kinase flux of zero or a small value.

With regard to the various alternatives, we have experimentally shown that malic enzyme activity is high in glucose-citrate-fed cultures; thus, the pathway incorporating the malic enzyme is more likely to be operational. However, we cannot at present assess the extent of HMP recycling from our experimental data. Experiments with radiolabeled glucose and citrate are planned to establish the degree of HMP recycling and the flux of malic enzyme.

Molecular Mechanism of Citrate Effects

Enzymes that exert significant control over the glycolytic flux are pyruvate kinase and PFK. We believe that activities of both enzymes are affected by citrate transport. Citrate transport is accompanied by divalent metal ions that increase the electroneu- trality during the uptake." Also, the transport system has a higher affinity for magnesium and manganese than calcium. The amount of citrate transported would require simultaneous transport and recycling of magnesium, manganese, and calcium present in the medium. Because calcium is a strong inhibitor of the enzyme pyruvate kinase in both prokaryoticll and eukaryotic cells,I0 its high intracellular levels could significantly inhibit pyruvate kinase activity and reduce the glycolytic flux. Moreover, inhibition of pyruvate kinase could lead to an elevated concentration of phosphoenolpyruvate (PEP). PEP is an effective inhibitor of PFK in bacteria? thus, reduction in PFK activity and hence further decline in the glycolytic flux is also likely. Another possibility of lower glycolytic flux in the presence of citrate is the direct inhibition of PFK by an elevated intracellular citrate concentration. Although


PFK is allosterically inhibited by citrate in higher cells,10 prokaryotic PFKs including those on E. coli and Bacillus stearothemophilus are not inhibited by citrate.’* Nonetheless, there is no information on B. subtilis PFK, its binding sites, and whether the enzyme is inhibited by citrate. We believe, however, that citrate inhibition of glycolytic flux through a series of events involving the inhibition of both pyruvatc kinase and PFK is more likely than the direct inhibition of PFK by citrate. Our recent intracellular measurements of PEP and other intermediate metabolites confirms this notion.12

Protein Productivity for Recombinant Bacillus Chemostats

B. subtilis has been used as a host system for protein production using rccombi- nant DNA t e ~ h n o l o g y . ~ ~ . ’ ~ Continuous culture techniques have been investigated for recombinant protein production, but low stability and productivity have been re- p ~ r t e d . ’ ~ - ~ ~ One significant problcm in continuous cultures of recombinant cells overexpressing a foreign protein is excessive acid formation.3x Because the technique of dual feeding was successfully used to reduce acid formation in the wild-type strain, extension of this technique was investigated for the recombinant system to investi- gate its effect on culture prod~ct ivi ty .~ An expression system consisting of the bacterium B. subtilis BR 151 carrying plasmid p602/19 was uscd. Thc studies were focused on the chemostat expression of CAT as a function of single- and dual- feeding strategies.

TABLE 8 summarizes a set of four chemostat runs with single and dual feed, at dilution rates of 0.2 and 0.4 hr-l. Acid formation was reduced threefold for the lower dilution rate for the dual-feed chemostat. Interestingly, the glucose-fed chemostat rcsulted in a washout at higher dilution rate, most probably due to excess acid formation, while in the dual-feed scenario, culture was stability maintained. It was found that in addition to the decrease in acid formation at D = 0.2 hr-l, cell density increased twofold and the CAT levels increased threefold (from 1000 U/mg protein to 3000 U/mg protein), which corresponds to a CAT level of about 12% of total cellular protein.’ This led to a sixfold increase in culture productivity. Because similar levels of CAT and cell densities were obtained for the higher dilution rate of 0.4 hr-’ for the dual-feed cultures, the overall culture productivity was further increased because of faster turnover of the reactor. Thus, the dual-feeding strategy was successful in reducing acid formation in recombinant cultures, and also resulted in a significant increase in culture productivity. To our knowledge, this is the first report of sustained and high levels of expression of recombinant proteins in bacterial chemostats.

TABLE 8. Results of Recombinant Chemostats for Various Feeds and Dilution Rates D SIC, S20 Slf Szr C Percent cut cut

S. No. (hr-I) (g/l) (gil) (gil) (g/l) Yield Acid spact” prod” 1 0.2 2 - 0 - 22 25 1000 35 2 0.4 2 - Wash Out -

3 0.2 2 0.64 0 0.49 38 9 3000 242 4 0.4 2 0.64 0 0.56 40 19 3000 420

- - - -

NOTE: Percent C yield is defined as (100 X g-carbon in cell/g carbon consumed from feed).

“CAT specific activity (U/mg protein). ’CAT productivity (1000 U/hr-I).

Percent acid is defined as (100 x g-carbon as acids/g carbon consumed from feed).


CONCLUSIONS

We hypothesized that if there was excess glycolytic flux, then instead of diverting the excess flux elsewhere, reducing the flux could potentially suppress acid production without adversely affecting growth rate. When B. subtilis cometabolizes glucose and citrate, the carbon yield reaches 0.73 g cell C/g total substrate carbon and no acid production results; the highest carbon yield is attained when the medium contains ca. 6 mol glucose/mol citrate. This indicates that the yield attainable when the negative impact of regulatory proccsses are minimized is high and approaches the theoretical yield, which ignores the dissipative processes. Finally, as a consequence of dual feeding, an order of magnitude increase in protein productivity was observed in the recombinant culture. This increased productivity was coupled with significant reduction in acid formation. Overall, these effects emerge from dual feeding by redistributing carbon flux such that the glycolytic flux is reduced and the degree of mismatch between glycolysis and TCA fluxes is also decreased. These beneficial effects also could result from a decreased pyruvate kinase activity, thereby eliminating the reliance on citrate co-feeding. Direct genetic alteration to eliminate the gene coding for the major pyruvate kinase activity is planned.

Optimal flux distribution and the effect of glucose-to-citrate feed ratio was also investigated from a network-optimization viewpoint. These analyses entailed identi- fying alternate flux distributions that have the high carbon yield corresponding to the glucose-to-citrate ratios examined in this study. The analysis revealed that optimal distribution of flux corresponds to lower glycolytic flux through PFK and pyruvate kinasexatalyzed reactions and indicated the possibility of recycling of the HMP pathway.

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38.

AcCoA CIT E4P F6P G6P KG MDH ME O M PEP PYR R5P Ribulose 5P succ TP Xylulose 5P 3PG

NOMENCLATURE

Abbreviations

acetyl coenzyme A citrate eryt hrose-4-phosphate fructose-6-phosphate glucose-6-phosphate a-ketoglutarate malate dehydrogenase malic enzyme oxaloacetate phosphoenol pyruvate pyruvate ribose-5-phosphate ribulose-5-phosphate succinate triose phosphate xylulose-5-phosphate 3-phosphoglycerate

Notation

A coefficient matrix Ti, r b constant vector YxlATP, Y,

particular flux or flux vector

ATP and carbon yield, respectively

Documents

Coordination of Glycolysis and TCA Cycle Reaction Networks