Analysis of Rate-limiting Reactions in Cephalosporin Biosynthesis" LI-HONG MALMBERG,b*CDAVID H. SHERMAN! AND WEI-SHOU Hub@ bDepartment of Chemical Engineering and Materials Science University of Minnesota Minneapolis, Minnesota 55455
dDepartment of Microbiology and Institute for Advanced Studies in Biological Process Technology University of Minnesota St. Paul, Minnesota 55108 INTRODUCTION
P-Lactam antibiotics, including penicillins and cephalosporins, are among the most important antimicrobial agents ever developed. Since their discovery decades ago, the productivity of these antibiotics has been improved by several orders of magnitude largely through strain improvement by means of mutagenesis. With the advent of recombinant DNA technology, there has been increasing interest in manipulating these biosynthetic pathways to improve the productivity further or to alter the product selectivity.The success of such genetic manipulations can be greatly facilitated if the rate-limiting enzymes or other controlling factors of the biosynthetic pathway can be identified. Many of the genes encoding the biosynthetic enzymes have been cloned and Among the better-studied organisms are Cephalosporium acremonium and Streptomyces clavuligems, which produce mostly cephalosporin C and cephamycin C, respectively. The early biosynthetic pathway for these antibiotics is shown in FIGURE1. First, three amino acid precursors, a-aminoadipic acid, cysteine, and valine, condense to form a tripeptide. After epimerization, the p-lactam ring is formed by a condensation reaction. The resulting isopenicillin N is subsequently converted to penicillin N by epimerase. A subsequent ring-expansion reaction converts the five-member ring to a sk-member ring, a characteristic of cephalosporin antibiotics. This reaction and the next hydroxylation reaction require oxygen and a cosubstrate, a-ketoglutarate. In S. clavuligems, the ring-expansion and hydroxylation reactions are catalyzed by two distinct enzymes, whereas, in C. acremonium, these two reactions are carried out by a bifunctional enzyme. After deacetylcephalosporin C, the reactions vary in S. clavuligems and in C. acremonium and eventually lead to the final products, cephamycin C and cephalosporin C, aThis work was partially supported by a grant from the National Science Foundation (No. ECE8552670). CLi-Hong Malmberg was supported by an NIGMS biotechnology training grant from the National Institutes of Health (No. GM 08347-01A1). eTo whom all correspondence should be addressed. 16
MALMBERG et al.: RATE-LIMITING REACTIONS
17
respectively. These final reactions have not been characterized in great detail. Lacking the kinetic data on the later reactions, we investigated the common reaction steps in these two microorganisms. Thus, our discussion and analysis will be restricted to the formation of deacetylcephalosporin C.
N H HOOC'
L z
~
~
-
(
~
~
NH SH z ) Z'CH-~H, l ~ ~ ~ CO OH'
L ~
Eplmonao
...
D
HOOC'CH'(CHz)l'CO"H
(PEN N) Expandaso
(DAOC)
0 2 , a-Ketoglutarate
OOH
Hydmxylaso
/ 0 2 , a-Ketoglutarato
FIGURE 1. The initial biosynthetic pathway of cephalosporins in C. acremonium and S. clavuligerus.
Recently, many attempts have been made to amplify the cloned genes of the biosynthetic pathway in the host cells. It was reported that the productivity of cephalosporin C was increased by amplifying the expandase/hydroxylase gene in C. acrernoniurn.6This is a successful example of the use of recombinant DNA methods
18
ANNALS NEW YORK ACADEMY OF SCIENCES
to improve p-lactam production. However, in order to effectively enhance productivity by genetic manipulation, identification of the controlling steps in the biosynthetic pathway is essential. Because the kinetic information on product formation and on enzyme activity is available, we performed a detailed kinetic analysis of the reaction steps. Here, we report our work and the identification of the rate-limiting steps involved in the cephalosporin biosynthesis in C. acremonium and S. clavuligems.
KINETIC ANALYSIS
The reaction rates of the biosynthetic steps can be represented by a set of first-order ordinary differential equations describing the concentration changes of the compounds involved in the pathway. These equations are as follows:
d [DAOC] - vexpandase - vhydroxylase - k[DAOC1, dt
(4)
d~[DAC] - vhydroxylase - vs - kLIDAC1, dt where [ACV], [IPN], [PenN], [DAOC], and [DAC] denote the intracellular concentrations of ACV tripeptide, isopenicillin N, penicillin N, deacetoxycephalosporin C, and deacetylcephalosporin C, respectively. VAcvs, VcyC~,,,, Vexpandase, and Vhydroxylase represent the rates of reactions catalyzed by ACV synthetase, cyclase, expandase, and V:pimerase are the forward and reverse and hydroxylase, respectively. VLpimerase reactions catalyzed by epimerase, respectively. V, is the rate of the subsequent reaction after the hydroxylation and methylation of the hydroxyl group in the case of S. clavuligerus or the acetyltransfer reaction in the case of C. acremonium. Last, k is the cell specific growth rate. In this analysis, we have considered only the initial reactions leading to deacetylcephalosporin C; the subsequent reaction rates are assumed to be relatively efficient such that the secretion rate of the final product is not restricted to these reactions
MALMBERG et 01.: RATE-LIMITING REACTIONS
19
and is dependent on the rate of hydroxylation. With this underlying assumption, the production rates of the final products, cephamycin C in S. clavuligetus and cephalosporin C in C. acremonium, are functions of the hydroxylation rate Vhydroxylase, the biomass concentration X , and the specific cell volume p:
where (Ceph) represents the extracellular concentration of cephalosporins in the culture medium. In the case of C. acremonium, a large amount of penicillin N is accumulated in the culture medium. Additional product formation is considered to account for penicillin N secretion into the culture medium as shown in equation 7. We assume that the secretion of penicillin N into the culture medium is controlled by simple diffusion caused by the concentration gradient across the cell surface as shown in equation 8: d ( PenN) ~dt
x‘P
’
VPenN,
VpenN= KSE.((PenN] - K . [PenN]),
(7)
(8)
where [PenN] and (PenN) denote the intracellular and extracellular concentrations of penicillin N; KSEand K represent the transport rate and partition coefficient, respectively . Cosubstrates, a-ketoglutarate, ATP, 02, and Fe+2are considered to be in excess and d o not limit the reaction steps. Single substrate reactions catalyzed by cyclase, epimerase, expandase, and hydroxylase have been characterized by simple MichaelisMenten kinetics for which the Michaelis constant K , and the maximum activity V,,, were measured:
The rate of the first reaction, nonribosomal condensation of ACV tripeptide, utilizing three substrates, a-aminoadipic acid, cysteine, and valine, is characterized by
where [A], [C], and [V] are the intracellular concentrations of a-aminoadipic acid, cysteine, and valine, respectively. KA, KC, and Kv denote the K , values for a-aminoadipic acid, cysteine, and valine, respectively. The Michaelis constants of the biosynthetic enzymes have been reported”” and are listed in TABLE1. Using the kinetic parameters shown in TABLE1 and the time profiles for maximum activities of enzymes during the batch culture reported by Zhang et al. ,14,1s shown in FIGURE 2, we simulated the specific production rate of deacetylcephalosporin C for S. clavuligerus
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TABLE1. Michaelis Constants of Cephalosporin Biosynthetic Enzymes in S. clavuligerus and C. acremoniuma S. clavuligencs
K,,,
Reference
C. acremonium K,,, Reference 0.17 7 0.026 7 0.34 7
Enzvme
Substrate
ACV synthetase
a-aminoadipic acid cysteine valine
0.56 0.07 1.14
10 10 10
cyclase
ACV tripeptide
0.32
11
epimerase
isopenicillin N penicillin N
0.30 0.78
12 12
expandase
penicillin N a-ketoglutarate
0.035 0.022
13 13
0.029 0.022
8 8
hydroxylase
DAOC a-ketoglutarate
0.029 0.022
13 13
0.020 0.022
8 8
0.34 -
9
-
“K, is in units of mM.
and C. acremonium during the batch culture. The results are shown in FIGURE3 and are compared to the specific production rates of cephamycin C by S. clavuligems and cephalosporin C by C. acremonium obtained from the experimental data reported by Zhang et al. 14,15 As can be seen, good agreements exist between the simulated and experimental results for both C. acremonium and S. clavuligerus. We subsequently performed a sensitivity analysis to evaluate the effect of the enzyme concentration on the production of cephalosporins. A sensitivity coefficient (Ci) of an enzyme is defined as the ratio of the relative change in the enzyme concentration to the relative change in the production rate of cephalosporins:
where Ei is the enzyme concentration, dEi is the perturbation in Ei, VCeph is the overall production rate of cephalosporins, and dVceph is the perturbation in VCeph. A sensitivity coefficient of 0 represents the case in which the level of the enzyme has no influence on the total output of cephalosporins. On the other hand, a sensitivity coefficient of 1 indicates that the enzyme has the total control of the specific production rate. Sensitivity coefficients for biosynthetic enzymes in both S. clavuligerus and C. acremonium were calculated theoretically. The sensitivity coefficients of enzymes in S. clavuligerus were constant throughout the fermentation and are shown in TABLE 2. ACV synthetase has the highest sensitivity coefficient, whereas the other enzymes have negligible influence on the production rate. Similar results are observed in the case of C. acremonium (FIGURE4); ACV synthetase exhibits the highest sensitivity coefficient among the biosynthetic enzymes. These results indicate that ACV synthetase is the rate-limiting enzyme in the biosynthesis of cephalosporins by S. clavuligerus and C. acrernonium.
MALMBERG el al.: RATE-LIMITING REACTIONS
21
EFFECT OF AMPLIFYING ENZYME LEVEL The effect of amplifying the rate-limiting enzyme, ACV synthetase, in S. clavuligems was examined theoretically. Results, shown in FIGURE 5 , indicate that increasing the ACV synthetase level leads to an increase in the production of cephalosporins. However, the amount of increase in production rate is not proportional to that of the enzyme level. This is because a second enzyme, hydroxylase, becomes rate-limiting as the ACV synthetase level increases. If both ACV synthetase and hydroxylase are amplified, the overall production rate increases linearly until a third enzyme becomes rate-limiting.
S. clavuligerus 3.0 2.5
i 1 Epirnerase
ACV Synlhetase h
40
80
60
100
120
140
TIME (HR)
C. acremonlum 2.5
--g c
-
I
I
15
2 -
0)
1.5
-
E > w
c
3
1 -
E
0.5
-
0
-'
0
1
hvdroxvlase . .
U
I
I
20
40
60
80
TIME
-0
100 120 140 160
(HR)
FIGURE 2. Maximum activity profiles of biosynthetic enzymes in S.cluvuligencs NRRL 3585 (upper figure)14 and in C. acremonium C-10 (lower figure)15 during the batch culture. O n e unit of enzyme activity represents one kmole of product formed per minute.
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In the production of cephalosporins by C. acremonium, a significant fraction of the antibiotics produced is penicillin N. By amplifying the enzyme, expandase, converting penicillin N to cephalosporin, it was shown that the accumulation of penicillin N in the medium was reduced.6 Using this kinetic model and the kinetic
2.0
,
0
clavuligerus
S.
20
60
40
80
100
120
140
TIME (HR)
C. ecremonlum
rc 0
.I
I
50
100
TIME
150
(HR)
FIGURE 3. Profiles of the simulated (-) and the experimental (-0-) specific production rate of cephalosporins during the batch culture in S. clavuligerus (upper figure) and C. acremonium (lower figure).
parameters described earlier, we examined the effect of amplifying expandase activity on the production of cephalosporin C. It should be noted that in C. acremonium the expandase and hydroxylase are a single bifunctional enzyme. Thus, the amplification effect is on both reaction steps. T h e results of such a simulation are
MALMBERG ef d.:RATE-LIMITING REACTIONS
23
TABLE 2. Sensitivity Coefficients of Biosynthetic Enzymes as Defined in Equation 11 Enzyme
Sensitivity Coefficient
ACV synthetase cyclase epimerase expandase hydroxylase
1.0 0.0 0.0 0.0 0.0
shown in FIGURE6. As can be seen, the beneficial effect of amplifying expandase alone is rather limited. A more profound beneficial effect can be obtained if both ACV synthetase and expandase are amplified.
DISCUSSION Through kinetic analysis, we have shown that cephalosporin biosynthesis in C. acremonium and S. clavuligenrs is limited by ACV synthetase. We have also shown that the beneficial effect of simply amplifying the rate-limiting enzyme is rather limited because a second enzyme could rapidly become the controlling factor. Throughout this analysis, we have made several assumptions in order to facilitate the analysis. These assumptions are as follows:
(1) ATP and a-ketoglutarate are in sufficient supply such that their concentrations are nearly at saturation and have no effect on the reaction; (2) the concentrations of the precursors remain constant throughout the cultivation period; (3) the kinetic data determined in vitro are applicable to the in vivo situation.
1.0
-
k! 0 IL
0.8
-
s
0.6
-
t
0.4
-
0.2
-
I-
z Y
>
2
c
z
v)
fn
0.0
20
I
I
I
I
1
1
40
60
80
100
120
140
TIME
(HR)
I 160
FIGURE 4. Profiles of sensitivity coefficients of biosynthetic enzymes during the batch culture in C. acremonium.
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/
ACV synthetase & hydroxylase
0
2
4
6
8
10
RELATIVE INCREASE IN ENZYME LEVEL
FIGURE 5. Specific production rate of cephalosporins as a function of the relative concentration of ACV synthetase (dashed line) or ACV synthetase and hydroxylase (solid line). The relative concentration at time t is defined as the ratio of the enzyme concentration to that shown in FIGURE 2 (upper) at time t .
Furthermore, to simplify our analysis, we also assumed that the precursor concentrations remained unchanged after amplification of the enzyme level as in the cases shown in FIGURES 5 and 6. These assumptions have not yet been verified and in some cases are probably not absolutely true. Nevertheless, with these assumptions, we are able to proceed with our analyses. The fact that the simulation results are consistent with the experimental data suggests that our kinetic model is reasonable in describing the biosynthesis of cephalosporins. Moreover, without these analyses, it is difficult to envision which enzyme(s) should be amplified by merely judging from
1 -
ACV synthetase & expandaselhydroxylase
expandaselhydroxylase
1
1 4 7 9 12 RELATIVE INCREASE IN ENZYME LEVEL
FIGURE 6. Specific production rate of cephalosporins as a function of the relative concentration of expandase/hydroxylase (dashed line) or ACV synthetase and expandase/hydroxylase (solid line). The relative concentration at time t is defined as the ratio of the enzyme 2 (lower) at time 1. concentration to that shown in FIGURE
MALMBERG et d.:RATE-LIMITINGREACTIONS
25
experimental measurement of enzyme activities. Such kinetic analysis also allows us to assess the beneficial effect of gene amplification. ACKNOWLEDGMENTS
We thank A. L. Demain for valuable discussions. REFERENCES
1. SAMSON,S. M., R. BELAGAJE,D. T. BLANKENSHIP, J. L. CHAPMAN, D. PERRY,P. L. SKATRUD, R. M. VANFRANK,E. P. ABRAHAM, J. E. BALDWIN, S. W. QUEENER & T. D. INGOLIA. 1985. Isolation, sequence determination, and expression in Escherichiu coli of the isopenicillin N synthetase gene from Cephalosporium ucremonium. Nature 3 1 8 191194. S. M., J. E. DOTZLAF,M. L. SLISZ,G. W. BECKER,R. M. VANFRANK, L. E. VEAL, 2. SAMSON, W-K. YEH, J. R. MILLER,S. W. QUEENER& T. D. INGOLIA.1987. Cloning and expression of the fungal expandase/hydroxylase gene involved in cephalosporin biosynthesis. Bio/Technology 5 1207-1214. S. WOLFE,L. C. VINING, D. W. S. M. MEVARECH, 3. LESKIW,B. K., Y. AHARONOWITZ, WESTLAKE & S. E. JENSEN. 1988. Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus. Gene 6 2 187-196. S., M. B. TOBIN& J. R. MILLER.1990. The p-lactam biosynthesis genes for 4. KOVACEVIC, isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus. J. Bacteriol. 172: 3952-3958. S., B. J. WEIGEL,M. B. TOBIN,T. D. INGOLIA & J. R. MILLER.1989. Cloning, 5. KOVACEVIC, characterization, and expression in Escherichiu coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J. Bacteriol. 171: 754-760. P. L., A. J. TIETZ,T. D. INGOLIA, C. A. CANTWELL, D. L. FISHER, J. L. CHAPMAN 6. SKATRUD, & S. W. QUEENER. 1989. Use of recombinant DNA to improve production of cephalosporin C by Cephalosporium acremonium. Bio/Technology 7: 477-485. & S. WOLFE.1987. 6-(L-a-Aminoadipyl)-L-cysteinyl-D-vaIine 7. BANKO,G., A. L. DEMAIN synthetase (ACV synthetase): a multifunctional enzyme with broad substrate specificity for the synthesis of penicillin and cephalosporin precursors. J. Am. Chem. SOC. 109 2858-2860. 8. DOTZLAF,J. E. & W-K. YEH.1987.Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephulosporium ucremonium. J. Bacteriol. 169 1611-1618. R. M. ADLINGTON, H-H. TING,R. L. WHITE,G. S. 9. PANG,C-P., B. CHAKRAVARTI, JAYATILAKE, J. E. BALDWIN & E. P. ABRAHAM. 1984. Purification of isopenicillin N synthetase. Biochem. J. 2 2 2 789-795. & S. WOLFE.1988. Production of the penicillin 10. JENSEN,S. E., D. W. S. WESTLAKE (ACV) by cell free extracts from precursor 6-(L-a-aminoadipyl)-L-cysteinyl-D-valine Streptomyces clavuligerus. FEMS Microbiol. Lett. 4 9 213-218. S. E., B. K. LESKIW, L. C. VINING,Y. AHARONOWITZ, D. W. S. WESTLAKE & S. 11. JENSEN, WOLFE.1986. Purification of isopenicillin N synthetase from Streptomyces clavuligerus. Can. J. Microbiol. 32: 953-958. 12. USUI,S. & C-A. Yu. 1989. Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus. Biochim. Biophys. Acta 999 78-85. & S. WOLFE. 1985. Deacetoxycephalosporin C 13. JENSEN,S. E., D. W. S. WESTLAKE synthetase and deacetoxycephalosporin C hydroxylase are two separate enzymes in Streptomyces clavuligerus. J. Antibiot. 3 8 263-265. 14. ZHANG,J., S. WOLFE& A. L. DEMAIN. 1989.Ammonium ions repress 6-(~-a-aminoadipyl)L-cysteinyl-D-valinesynthetase in Srreptomyces clavuligerus NRRL 3585. Can. J. Microbiol. 35: 399-402.
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15. ZHANG,J., S. WOLFE& A. L. DEMAIN. 1987. Effect of ammonium as nitrogen source on production of 6-(L-cu-aminoadipyl)-L-cysteinyl-D-valine synthetase by Cephalosporium acremonium C-10. J. Antibiot. (Tokyo)4 0 1746-1750.
APPENDIX Nomenclature A, C, V ACV
Ceph DAC DAOC IPN K Km
KSE
PenN S V vmax
X CL
P
[I
I)
a-aminoadipic acid, cysteine, valine 6-( L-a-aminoadipyl)-L-cysteinyl-D-valine cephalosporins deacetylcephalosporin C deacetoxycephalosporin C isopenicillin N partition coefficient Michaelis constant (mM) transport rate (l/min) penicillin N substrate reaction rate (pmol/min/pL cell volume) maximum velocity of enzyme (pmol/min/kL cell volume) biomass concentration (mg dry cell weight/mL culture) specific growth rate specific cell volume (FL cell volume/mg dry cell weight) intracellular concentration (nmol/FL cell volume) extracellular concentration (nmol/mL culture)
dDepartment of Microbiology and Institute for Advanced Studies in Biological Process Technology University of Minnesota St. Paul, Minnesota 55108 INTRODUCTION
P-Lactam antibiotics, including penicillins and cephalosporins, are among the most important antimicrobial agents ever developed. Since their discovery decades ago, the productivity of these antibiotics has been improved by several orders of magnitude largely through strain improvement by means of mutagenesis. With the advent of recombinant DNA technology, there has been increasing interest in manipulating these biosynthetic pathways to improve the productivity further or to alter the product selectivity.The success of such genetic manipulations can be greatly facilitated if the rate-limiting enzymes or other controlling factors of the biosynthetic pathway can be identified. Many of the genes encoding the biosynthetic enzymes have been cloned and Among the better-studied organisms are Cephalosporium acremonium and Streptomyces clavuligems, which produce mostly cephalosporin C and cephamycin C, respectively. The early biosynthetic pathway for these antibiotics is shown in FIGURE1. First, three amino acid precursors, a-aminoadipic acid, cysteine, and valine, condense to form a tripeptide. After epimerization, the p-lactam ring is formed by a condensation reaction. The resulting isopenicillin N is subsequently converted to penicillin N by epimerase. A subsequent ring-expansion reaction converts the five-member ring to a sk-member ring, a characteristic of cephalosporin antibiotics. This reaction and the next hydroxylation reaction require oxygen and a cosubstrate, a-ketoglutarate. In S. clavuligems, the ring-expansion and hydroxylation reactions are catalyzed by two distinct enzymes, whereas, in C. acremonium, these two reactions are carried out by a bifunctional enzyme. After deacetylcephalosporin C, the reactions vary in S. clavuligems and in C. acremonium and eventually lead to the final products, cephamycin C and cephalosporin C, aThis work was partially supported by a grant from the National Science Foundation (No. ECE8552670). CLi-Hong Malmberg was supported by an NIGMS biotechnology training grant from the National Institutes of Health (No. GM 08347-01A1). eTo whom all correspondence should be addressed. 16
MALMBERG et al.: RATE-LIMITING REACTIONS
17
respectively. These final reactions have not been characterized in great detail. Lacking the kinetic data on the later reactions, we investigated the common reaction steps in these two microorganisms. Thus, our discussion and analysis will be restricted to the formation of deacetylcephalosporin C.
N H HOOC'
L z
~
~
-
(
~
~
NH SH z ) Z'CH-~H, l ~ ~ ~ CO OH'
L ~
Eplmonao
...
D
HOOC'CH'(CHz)l'CO"H
(PEN N) Expandaso
(DAOC)
0 2 , a-Ketoglutarate
OOH
Hydmxylaso
/ 0 2 , a-Ketoglutarato
FIGURE 1. The initial biosynthetic pathway of cephalosporins in C. acremonium and S. clavuligerus.
Recently, many attempts have been made to amplify the cloned genes of the biosynthetic pathway in the host cells. It was reported that the productivity of cephalosporin C was increased by amplifying the expandase/hydroxylase gene in C. acrernoniurn.6This is a successful example of the use of recombinant DNA methods
18
ANNALS NEW YORK ACADEMY OF SCIENCES
to improve p-lactam production. However, in order to effectively enhance productivity by genetic manipulation, identification of the controlling steps in the biosynthetic pathway is essential. Because the kinetic information on product formation and on enzyme activity is available, we performed a detailed kinetic analysis of the reaction steps. Here, we report our work and the identification of the rate-limiting steps involved in the cephalosporin biosynthesis in C. acremonium and S. clavuligems.
KINETIC ANALYSIS
The reaction rates of the biosynthetic steps can be represented by a set of first-order ordinary differential equations describing the concentration changes of the compounds involved in the pathway. These equations are as follows:
d [DAOC] - vexpandase - vhydroxylase - k[DAOC1, dt
(4)
d~[DAC] - vhydroxylase - vs - kLIDAC1, dt where [ACV], [IPN], [PenN], [DAOC], and [DAC] denote the intracellular concentrations of ACV tripeptide, isopenicillin N, penicillin N, deacetoxycephalosporin C, and deacetylcephalosporin C, respectively. VAcvs, VcyC~,,,, Vexpandase, and Vhydroxylase represent the rates of reactions catalyzed by ACV synthetase, cyclase, expandase, and V:pimerase are the forward and reverse and hydroxylase, respectively. VLpimerase reactions catalyzed by epimerase, respectively. V, is the rate of the subsequent reaction after the hydroxylation and methylation of the hydroxyl group in the case of S. clavuligerus or the acetyltransfer reaction in the case of C. acremonium. Last, k is the cell specific growth rate. In this analysis, we have considered only the initial reactions leading to deacetylcephalosporin C; the subsequent reaction rates are assumed to be relatively efficient such that the secretion rate of the final product is not restricted to these reactions
MALMBERG et 01.: RATE-LIMITING REACTIONS
19
and is dependent on the rate of hydroxylation. With this underlying assumption, the production rates of the final products, cephamycin C in S. clavuligetus and cephalosporin C in C. acremonium, are functions of the hydroxylation rate Vhydroxylase, the biomass concentration X , and the specific cell volume p:
where (Ceph) represents the extracellular concentration of cephalosporins in the culture medium. In the case of C. acremonium, a large amount of penicillin N is accumulated in the culture medium. Additional product formation is considered to account for penicillin N secretion into the culture medium as shown in equation 7. We assume that the secretion of penicillin N into the culture medium is controlled by simple diffusion caused by the concentration gradient across the cell surface as shown in equation 8: d ( PenN) ~dt
x‘P
’
VPenN,
VpenN= KSE.((PenN] - K . [PenN]),
(7)
(8)
where [PenN] and (PenN) denote the intracellular and extracellular concentrations of penicillin N; KSEand K represent the transport rate and partition coefficient, respectively . Cosubstrates, a-ketoglutarate, ATP, 02, and Fe+2are considered to be in excess and d o not limit the reaction steps. Single substrate reactions catalyzed by cyclase, epimerase, expandase, and hydroxylase have been characterized by simple MichaelisMenten kinetics for which the Michaelis constant K , and the maximum activity V,,, were measured:
The rate of the first reaction, nonribosomal condensation of ACV tripeptide, utilizing three substrates, a-aminoadipic acid, cysteine, and valine, is characterized by
where [A], [C], and [V] are the intracellular concentrations of a-aminoadipic acid, cysteine, and valine, respectively. KA, KC, and Kv denote the K , values for a-aminoadipic acid, cysteine, and valine, respectively. The Michaelis constants of the biosynthetic enzymes have been reported”” and are listed in TABLE1. Using the kinetic parameters shown in TABLE1 and the time profiles for maximum activities of enzymes during the batch culture reported by Zhang et al. ,14,1s shown in FIGURE 2, we simulated the specific production rate of deacetylcephalosporin C for S. clavuligerus
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TABLE1. Michaelis Constants of Cephalosporin Biosynthetic Enzymes in S. clavuligerus and C. acremoniuma S. clavuligencs
K,,,
Reference
C. acremonium K,,, Reference 0.17 7 0.026 7 0.34 7
Enzvme
Substrate
ACV synthetase
a-aminoadipic acid cysteine valine
0.56 0.07 1.14
10 10 10
cyclase
ACV tripeptide
0.32
11
epimerase
isopenicillin N penicillin N
0.30 0.78
12 12
expandase
penicillin N a-ketoglutarate
0.035 0.022
13 13
0.029 0.022
8 8
hydroxylase
DAOC a-ketoglutarate
0.029 0.022
13 13
0.020 0.022
8 8
0.34 -
9
-
“K, is in units of mM.
and C. acremonium during the batch culture. The results are shown in FIGURE3 and are compared to the specific production rates of cephamycin C by S. clavuligems and cephalosporin C by C. acremonium obtained from the experimental data reported by Zhang et al. 14,15 As can be seen, good agreements exist between the simulated and experimental results for both C. acremonium and S. clavuligerus. We subsequently performed a sensitivity analysis to evaluate the effect of the enzyme concentration on the production of cephalosporins. A sensitivity coefficient (Ci) of an enzyme is defined as the ratio of the relative change in the enzyme concentration to the relative change in the production rate of cephalosporins:
where Ei is the enzyme concentration, dEi is the perturbation in Ei, VCeph is the overall production rate of cephalosporins, and dVceph is the perturbation in VCeph. A sensitivity coefficient of 0 represents the case in which the level of the enzyme has no influence on the total output of cephalosporins. On the other hand, a sensitivity coefficient of 1 indicates that the enzyme has the total control of the specific production rate. Sensitivity coefficients for biosynthetic enzymes in both S. clavuligerus and C. acremonium were calculated theoretically. The sensitivity coefficients of enzymes in S. clavuligerus were constant throughout the fermentation and are shown in TABLE 2. ACV synthetase has the highest sensitivity coefficient, whereas the other enzymes have negligible influence on the production rate. Similar results are observed in the case of C. acremonium (FIGURE4); ACV synthetase exhibits the highest sensitivity coefficient among the biosynthetic enzymes. These results indicate that ACV synthetase is the rate-limiting enzyme in the biosynthesis of cephalosporins by S. clavuligerus and C. acrernonium.
MALMBERG el al.: RATE-LIMITING REACTIONS
21
EFFECT OF AMPLIFYING ENZYME LEVEL The effect of amplifying the rate-limiting enzyme, ACV synthetase, in S. clavuligems was examined theoretically. Results, shown in FIGURE 5 , indicate that increasing the ACV synthetase level leads to an increase in the production of cephalosporins. However, the amount of increase in production rate is not proportional to that of the enzyme level. This is because a second enzyme, hydroxylase, becomes rate-limiting as the ACV synthetase level increases. If both ACV synthetase and hydroxylase are amplified, the overall production rate increases linearly until a third enzyme becomes rate-limiting.
S. clavuligerus 3.0 2.5
i 1 Epirnerase
ACV Synlhetase h
40
80
60
100
120
140
TIME (HR)
C. acremonlum 2.5
--g c
-
I
I
15
2 -
0)
1.5
-
E > w
c
3
1 -
E
0.5
-
0
-'
0
1
hvdroxvlase . .
U
I
I
20
40
60
80
TIME
-0
100 120 140 160
(HR)
FIGURE 2. Maximum activity profiles of biosynthetic enzymes in S.cluvuligencs NRRL 3585 (upper figure)14 and in C. acremonium C-10 (lower figure)15 during the batch culture. O n e unit of enzyme activity represents one kmole of product formed per minute.
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In the production of cephalosporins by C. acremonium, a significant fraction of the antibiotics produced is penicillin N. By amplifying the enzyme, expandase, converting penicillin N to cephalosporin, it was shown that the accumulation of penicillin N in the medium was reduced.6 Using this kinetic model and the kinetic
2.0
,
0
clavuligerus
S.
20
60
40
80
100
120
140
TIME (HR)
C. ecremonlum
rc 0
.I
I
50
100
TIME
150
(HR)
FIGURE 3. Profiles of the simulated (-) and the experimental (-0-) specific production rate of cephalosporins during the batch culture in S. clavuligerus (upper figure) and C. acremonium (lower figure).
parameters described earlier, we examined the effect of amplifying expandase activity on the production of cephalosporin C. It should be noted that in C. acremonium the expandase and hydroxylase are a single bifunctional enzyme. Thus, the amplification effect is on both reaction steps. T h e results of such a simulation are
MALMBERG ef d.:RATE-LIMITING REACTIONS
23
TABLE 2. Sensitivity Coefficients of Biosynthetic Enzymes as Defined in Equation 11 Enzyme
Sensitivity Coefficient
ACV synthetase cyclase epimerase expandase hydroxylase
1.0 0.0 0.0 0.0 0.0
shown in FIGURE6. As can be seen, the beneficial effect of amplifying expandase alone is rather limited. A more profound beneficial effect can be obtained if both ACV synthetase and expandase are amplified.
DISCUSSION Through kinetic analysis, we have shown that cephalosporin biosynthesis in C. acremonium and S. clavuligenrs is limited by ACV synthetase. We have also shown that the beneficial effect of simply amplifying the rate-limiting enzyme is rather limited because a second enzyme could rapidly become the controlling factor. Throughout this analysis, we have made several assumptions in order to facilitate the analysis. These assumptions are as follows:
(1) ATP and a-ketoglutarate are in sufficient supply such that their concentrations are nearly at saturation and have no effect on the reaction; (2) the concentrations of the precursors remain constant throughout the cultivation period; (3) the kinetic data determined in vitro are applicable to the in vivo situation.
1.0
-
k! 0 IL
0.8
-
s
0.6
-
t
0.4
-
0.2
-
I-
z Y
>
2
c
z
v)
fn
0.0
20
I
I
I
I
1
1
40
60
80
100
120
140
TIME
(HR)
I 160
FIGURE 4. Profiles of sensitivity coefficients of biosynthetic enzymes during the batch culture in C. acremonium.
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/
ACV synthetase & hydroxylase
0
2
4
6
8
10
RELATIVE INCREASE IN ENZYME LEVEL
FIGURE 5. Specific production rate of cephalosporins as a function of the relative concentration of ACV synthetase (dashed line) or ACV synthetase and hydroxylase (solid line). The relative concentration at time t is defined as the ratio of the enzyme concentration to that shown in FIGURE 2 (upper) at time t .
Furthermore, to simplify our analysis, we also assumed that the precursor concentrations remained unchanged after amplification of the enzyme level as in the cases shown in FIGURES 5 and 6. These assumptions have not yet been verified and in some cases are probably not absolutely true. Nevertheless, with these assumptions, we are able to proceed with our analyses. The fact that the simulation results are consistent with the experimental data suggests that our kinetic model is reasonable in describing the biosynthesis of cephalosporins. Moreover, without these analyses, it is difficult to envision which enzyme(s) should be amplified by merely judging from
1 -
ACV synthetase & expandaselhydroxylase
expandaselhydroxylase
1
1 4 7 9 12 RELATIVE INCREASE IN ENZYME LEVEL
FIGURE 6. Specific production rate of cephalosporins as a function of the relative concentration of expandase/hydroxylase (dashed line) or ACV synthetase and expandase/hydroxylase (solid line). The relative concentration at time t is defined as the ratio of the enzyme 2 (lower) at time 1. concentration to that shown in FIGURE
MALMBERG et d.:RATE-LIMITINGREACTIONS
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experimental measurement of enzyme activities. Such kinetic analysis also allows us to assess the beneficial effect of gene amplification. ACKNOWLEDGMENTS
We thank A. L. Demain for valuable discussions. REFERENCES
1. SAMSON,S. M., R. BELAGAJE,D. T. BLANKENSHIP, J. L. CHAPMAN, D. PERRY,P. L. SKATRUD, R. M. VANFRANK,E. P. ABRAHAM, J. E. BALDWIN, S. W. QUEENER & T. D. INGOLIA. 1985. Isolation, sequence determination, and expression in Escherichiu coli of the isopenicillin N synthetase gene from Cephalosporium ucremonium. Nature 3 1 8 191194. S. M., J. E. DOTZLAF,M. L. SLISZ,G. W. BECKER,R. M. VANFRANK, L. E. VEAL, 2. SAMSON, W-K. YEH, J. R. MILLER,S. W. QUEENER& T. D. INGOLIA.1987. Cloning and expression of the fungal expandase/hydroxylase gene involved in cephalosporin biosynthesis. Bio/Technology 5 1207-1214. S. WOLFE,L. C. VINING, D. W. S. M. MEVARECH, 3. LESKIW,B. K., Y. AHARONOWITZ, WESTLAKE & S. E. JENSEN. 1988. Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus. Gene 6 2 187-196. S., M. B. TOBIN& J. R. MILLER.1990. The p-lactam biosynthesis genes for 4. KOVACEVIC, isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus. J. Bacteriol. 172: 3952-3958. S., B. J. WEIGEL,M. B. TOBIN,T. D. INGOLIA & J. R. MILLER.1989. Cloning, 5. KOVACEVIC, characterization, and expression in Escherichiu coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J. Bacteriol. 171: 754-760. P. L., A. J. TIETZ,T. D. INGOLIA, C. A. CANTWELL, D. L. FISHER, J. L. CHAPMAN 6. SKATRUD, & S. W. QUEENER. 1989. Use of recombinant DNA to improve production of cephalosporin C by Cephalosporium acremonium. Bio/Technology 7: 477-485. & S. WOLFE.1987. 6-(L-a-Aminoadipyl)-L-cysteinyl-D-vaIine 7. BANKO,G., A. L. DEMAIN synthetase (ACV synthetase): a multifunctional enzyme with broad substrate specificity for the synthesis of penicillin and cephalosporin precursors. J. Am. Chem. SOC. 109 2858-2860. 8. DOTZLAF,J. E. & W-K. YEH.1987.Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephulosporium ucremonium. J. Bacteriol. 169 1611-1618. R. M. ADLINGTON, H-H. TING,R. L. WHITE,G. S. 9. PANG,C-P., B. CHAKRAVARTI, JAYATILAKE, J. E. BALDWIN & E. P. ABRAHAM. 1984. Purification of isopenicillin N synthetase. Biochem. J. 2 2 2 789-795. & S. WOLFE.1988. Production of the penicillin 10. JENSEN,S. E., D. W. S. WESTLAKE (ACV) by cell free extracts from precursor 6-(L-a-aminoadipyl)-L-cysteinyl-D-valine Streptomyces clavuligerus. FEMS Microbiol. Lett. 4 9 213-218. S. E., B. K. LESKIW, L. C. VINING,Y. AHARONOWITZ, D. W. S. WESTLAKE & S. 11. JENSEN, WOLFE.1986. Purification of isopenicillin N synthetase from Streptomyces clavuligerus. Can. J. Microbiol. 32: 953-958. 12. USUI,S. & C-A. Yu. 1989. Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus. Biochim. Biophys. Acta 999 78-85. & S. WOLFE. 1985. Deacetoxycephalosporin C 13. JENSEN,S. E., D. W. S. WESTLAKE synthetase and deacetoxycephalosporin C hydroxylase are two separate enzymes in Streptomyces clavuligerus. J. Antibiot. 3 8 263-265. 14. ZHANG,J., S. WOLFE& A. L. DEMAIN. 1989.Ammonium ions repress 6-(~-a-aminoadipyl)L-cysteinyl-D-valinesynthetase in Srreptomyces clavuligerus NRRL 3585. Can. J. Microbiol. 35: 399-402.
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15. ZHANG,J., S. WOLFE& A. L. DEMAIN. 1987. Effect of ammonium as nitrogen source on production of 6-(L-cu-aminoadipyl)-L-cysteinyl-D-valine synthetase by Cephalosporium acremonium C-10. J. Antibiot. (Tokyo)4 0 1746-1750.
APPENDIX Nomenclature A, C, V ACV
Ceph DAC DAOC IPN K Km
KSE
PenN S V vmax
X CL
P
[I
I)
a-aminoadipic acid, cysteine, valine 6-( L-a-aminoadipyl)-L-cysteinyl-D-valine cephalosporins deacetylcephalosporin C deacetoxycephalosporin C isopenicillin N partition coefficient Michaelis constant (mM) transport rate (l/min) penicillin N substrate reaction rate (pmol/min/pL cell volume) maximum velocity of enzyme (pmol/min/kL cell volume) biomass concentration (mg dry cell weight/mL culture) specific growth rate specific cell volume (FL cell volume/mg dry cell weight) intracellular concentration (nmol/FL cell volume) extracellular concentration (nmol/mL culture)
