* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Metabolic Flux Analysis on the Production of Poly(3 - Wiley-VCH
Survey
Document related concepts
Biochemical cascade wikipedia , lookup
Butyric acid wikipedia , lookup
Microbial metabolism wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
Fatty acid metabolism wikipedia , lookup
Metabolomics wikipedia , lookup
Biochemistry wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Pharmacometabolomics wikipedia , lookup
Biosynthesis wikipedia , lookup
Basal metabolic rate wikipedia , lookup
Isotopic labeling wikipedia , lookup
Citric acid cycle wikipedia , lookup
Transcript
249 8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate) Prof. Dr. Sang Yup Lee1, M. Eng. Soon Ho Hong2, M. Eng. Si Jae Park3, Dr. Richard van Wegen4, Dr. Anton P. J. Middelberg5 1 Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea; Tel.: 82-42-869-3930; Fax: 82-42-869-3910; E-mail: [email protected] 2 Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea; Tel.: 82-42-869-5970; Fax: 82-42-869-3910; E-mail: [email protected] 3 Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea; Tel.: 82-42-869-5970; Fax: 82-42-869-3910; E-mail: [email protected] 4 Department of Chemical Engineering, University of Adelaide, SA 5005, Australia; Tel.: 43(01) 796-6362-311; Fax: 43(01) 796-6362-333; E-mail: [email protected] 5 Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK; Tel.: 44-1223-335245; Fax: 44-1223-334796; E-mail: [email protected] 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 2 Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 3 Production of Poly(3HB ) by Ralstonia eutropha . . . . . . . . . . . . . . . . . . 252 4 Production of Poly(3HB ) by Recombinant Escherichia coli . . . . . . . . . . . . 255 5 Conclusions and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 250 8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate) 6 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 3HB CoA DOC MCA MFA NADH NADPH PHA SCL TCA 3-hydroxybutyrate coenzyme A dissolved oxygen concentration metabolic control analysis metabolic flux analysis nicotinamide adenine dinucleotide (reduced form) nicotinamide adenine dinucleotide phosphate (reduced form) polyhydroxyalkanoate short-chain-length tricarboxylic acid cycle 1 Introduction Polyhydroxyalkanoates (PHAs) have been considered to be good candidates as alternatives to synthetic nondegradable polymers due to their similar mechanical properties to petroleum-derived polymers and their complete biodegradability (Steinb¸chel, 1992; Brandl et al., 1995; Lee, 1996a). A number of microorganisms have been found to accumulate PHAs under unfavorable growth conditions and in the presence of excess carbon source (Anderson and Dawes, 1990; Doi, 1990; Steinb¸chel, 1991; Lee, 1996b; Steinb¸chel and F¸chtenbusch, 1998; Madison and Huisman, 1999). Among the various members of PHAs, poly(3-hydroxybutyrate), poly(3HB ), a member of shortchain-length (SCL ) PHAs, is best characterized and has been produced on a semicommercial scale. One of the major drawbacks in the commercialization of PHAs is the much higher production cost of PHAs compared with petrochemical-based polymers. Therefore, much effort has been devoted to reduce the production costs of PHAs by developing better bacterial strains and downstream processes such as more efficient fermentation and more economical recovery processes (Lee, 1996a,b; Choi et al., 1998; Choi and Lee, 1999a,b). Process design and economic analysis of SCL-PHA production by various bacteria have been reported, which provided the guidelines for designing an efficient means of PHA production (Choi and Lee, 1997, 1999c, 2000; Lee and Choi, 1998). Several factors affecting the production cost of PHA, including PHA productivity, PHA content and yield on carbon substrate, the cost of carbon substrate, and the recovery yield of PHA have been examined in detail. Based on the results of economic evaluation, Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli have been suggested as good candidates for the efficient production of SCL-PHAs (Lee, 1996a,b). By using a metabolic flux analysis (MFA ) technique, the intracellular metabolic fluxes can be quantified by the measurement of extracellular metabolite concentrations in 2 Historical Outline combination with the stoichiometry of intracellular reactions (Nielsen and Villadsen, 1994; Edwards et al., 1999). MFA is based on the pseudo-steady-state assumption, which means that there is no net accumulation of intermediates (Stephanopoulos, 1999). To analyze the system using the MFA technique, the system should be determined or over-determined, which means that the number of constraints is equal to or greater than that of the reactions. To analyze the under-determined system, more constraints are required. This is often replaced by setting up an objective function, such as maximum growth or maximum metabolite production, and solving by linear programing ( Varma and Palsson, 1994). MFA has been applied to calculate the maximum theoretical yield of a desired metabolite to be produced. Another application is to identify the rigidity of branch points in metabolic pathways. The rigidity of a branch point is important, as a rigid branch point resists changes in flux split ratios, while a flexible branch point tends to be more accommodating (Stephanopoulos and Vallino, 1991). The third possible application is the identification of alternative metabolic pathways. The detailed theories and applications of the MFA can be found in recent reviews (Edwards et al., 1999; Stephanopoulos, 1999). Metabolic control analysis (MCA ) is a statistical modeling technique that can be used to understand the control of metabolic pathways and pathway regulations. MCA allows us to understand how metabolic fluxes are controlled by certain enzyme activities and metabolite concentrations (Kacser and Burns, 1973; Heinrich and Rapoport, 1974). The responses of small changes in enzyme activities and metabolite concentrations to metabolic flux distribution can be predicted by MCA (Nielsen and Villadsen, 1994). If we consider a linear chain of N enzymatic reactions, there are N 1 intermediates Xj, j 1, º, N 1. The response coefficient of a certain metabolite is defined as the ratio of the relative change in the reaction rate brought about by the change in the metabolite concentration, viz: eji Xj @ri ; ri @Xj i 1; ::; N and j 1; ::; N 1 where ri is the net rate of the i-th enzymatic reaction and Xj is the size of the j-th metabolite pool. The flux control coefficient of a certain enzyme is the relative change in the steady-state flux resulting from the change in the activity of an enzyme of the pathway, viz: Cir Xei @r ; r @Xei i 1; :::; N where Xei is the activity of i-th enzyme and r is the overall steady-state flux. By analyzing the flux control coefficients and response coefficients, one can propose which enzymatic reaction step is rate controlling (e.g., a reaction step with high flux control coefficient is the rate-controlling step), and also predict the results of deviations in certain enzymatic reactions. Readers are encouraged to refer to an excellent monograph on MCA by Fell (1997). In this chapter, we review the applications of MFA and MCA on the production of poly(3HB ) by various bacterial strains. The effects of various environmental conditions on poly(3HB ) production are evaluated, and the important factors such as intracellular metabolite concentrations and enzyme activities on poly(3HB ) biosynthesis are examined by MFA and MCA. 2 Historical Outline A brief historical outline of the commercial production of poly(3HB ) is shown below: 251 252 8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate) Year History Strain 1925 Production of poly(3HB) Bacillus first discovered megaterium 1973 MCA first proposed 1988 The first phb full operon Ralstonia cloned eutropha 1988 Poly(3HB) first proEscherichia duced in E. coli coli 1991 Metabolic engineering first proposed 1993 Effect of nitrogen limRalstonia itation analyzed eutropha 1997 Effect of carbon sources Ralstonia analyzed eutropha 1998 Poly(3HB)-producing mechanism identified 1999 Effect of oxygen limita- Escherichia tion analyzed coli 2001 Control factors of poEscherichia ly(3HB) production coli identified 3 Production of Poly(3HB) by Ralstonia eutropha Ralstonia eutropha (formerly Alcaligenes eutrophus), has been intensively examined for the efficient production of SCL-PHAs. The PHA biosynthetic pathway in R. eutropha has been well characterized: two acetyl-CoA moieties are condensed to form acetoacetyl-CoA by b-ketothiolase. AcetoacetylCoA is then reduced to (R )-3-hydroxybutyryl-CoA by an NADPH-dependent reductase. PHA synthase finally links (R )-3hydroxybutyryl-CoA to the growing chain of poly(3HB ). The genes coding for the three enzymes were found to form a phb operon in the order of PHA synthase, b-ketothiolase, and reductase (Schubert et al., 1988; Slater et al., 1988; Peoples and Sinskey, 1989). The three-step poly(3HB ) biosynthesis pathway from acetyl-CoA is shown in Figure 1. Fedbatch culture strategies to achieve high productivity of PHAs have also been developed (Kim et al., 1994a,b; Ryu et al., 1997). An optimal fed-batch culture strategy resulted in both high poly(3HB ) concentration and productivity of 232 g L 1 and 3.14 g poly(3HB ) L 1 h 1, respectively (Ryu et al., 1997). The cost of raw materials (especially of the carbon source) is a very important factor affecting the overall production cost of poly(3HB ). Therefore, the effect of different carbon sources on the production of poly(3HB ) was extensively analyzed using MFA (Shi et al., 1997). R. eutropha can utilize various organic acids as carbon sources which, from a practical stand-point, is important for the production of poly(3HB ) from organic wastes, for example food waste. Consequently, butyrate, lactate, and acetate were examined as carbon sources. When R. eutropha was cultivated on a mixture of three carbon sources, lactate was consumed first as a large amount of ATP is needed for the transport of acetate and butyrate. The central metabolic pathways for the utilization of three carbon sources are very similar, except for the anaplerotic pathway which replenishes carbon intermediates to the tricarboxylic acid (TCA ) cycle. As shown in Figure 2, carbon dioxide is wasted during the conversion of pyruvate to acetyl-CoA when lactate is used as a carbon source. To decide which carbon source is better for the production of poly(3HB ), simulations were carried out at different specific growth rates with three carbon sources. The maximum poly(3HB ) yields (g g 1) without cell growth obtainable with acetate, lactate, and butyrate were 0.33, 0.33, and 0.67, respectively. The poly(3HB ) yield was limited by NADPH regeneration, as it is required for the conversion of acetoacetyl-CoA to (R )-3- 3 Production of Poly(3HB) by Ralstonia eutropha Fig. 1 Simplified central metabolic pathway of E. coli and PHB biosynthesis pathway. hydroxybutyryl-CoA. NADPH is regenerated solely by isocitrate dehydrogenase in the TCA cycle. Butyrate was found to be more efficient for the production of poly(3HB ), because about 67% of butyrate was directed to the TCA cycle, in which the required NADPH was regenerated, while only 33% of acetate and lactate were metabolized through the TCA cycle (Figure 2B, C, and D ). Poly(3HB ) production by R. eutropha H16 (ATCC 17699) was also examined by fedbatch cultivation using butyrate as a carbon source (Shimizu et al., 1993). The maximum poly(3HB ) yield of 0.85 (gg 1) was obtained at a butyrate concentration of 3 g L 1 and at pH 8.0. The final poly(3HB) content was 75 wt.% dry cell weight. The changing profiles of the intracellular metabolic flux distribution during the fermentation were evaluated by MFA (Shi et al., 1997). The accumulation of poly(3HB) was induced by depletion of the nitrogen source. When the strain was shifted from the growth phase to the poly(3HB ) production phase, flux into the poly(3HB) biosynthetic pathway increased, whilst flux into the glyoxylate bypass decreased (Figure 2A, B ). There was no significant change in flux to the TCA cycle through 253 254 8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate) Fig. 2 Flux distribution during fed-batch culture on (A ) butyrate at cell growth phase and (B ) at poly(3HB) production phase, and on (C ) acetate and (D ) lactate at poly(3HB) production phase. Fluxes were normalized by substrates uptake rates. [ Redrawn from Shi et al. (1997) with permission.] isocitrate dehydrogenase. It can be concluded that glyoxylate bypass is required for the growth on butyrate, and this competes with the poly(3HB ) biosynthetic pathway. Since flux into the TCA cycle was almost constant throughout the cultivation, the amount of NADPH produced during cultivation was not significantly changed. The NADPH consumption rate, however, was decreased because limitation of the nitrogen source blocked the amino acid synthesis pathways, especially the reaction from a-ketoglutarate to glutamate, which assimilates ammonium ions into the cell. Therefore, the residual NADPH was accumulated, which in turn enhanced poly(3HB ) biosynthesis. As mentioned earlier, the cost of raw materials is one of the most important factors affecting the economics of PHA production. Since the cost of glucose is lower than that of butyrate, glucose is better for the industrial production of PHAs. To 4 Production of Poly(3HB) by Recombinant Escherichia coli use glucose directly as a carbon source, a mixed culture system was examined, in which glucose was converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly(3HB ) by R. eutropha (Katoh et al., 1999). To evaluate the effects of environmental conditions on cell growth and poly(3HB ) production, MFA was carried out under various conditions. It was found that NH3 plays an important role in the production of poly(3HB ). When NH3 was sufficient, most of the NADPH was utilized for amino acids biosynthesis, and cell growth was activated. NADPH was used as a coenzyme of acetoacetyl-CoA reductase for the conversion of acetoacetyl-CoA to (R )-3hydroxybutyryl-CoA under nitrogen-limiting conditions. From these results, it can be said that poly(3HB ) production can be enhanced by providing a condition in which NADPH is in excess. 4 Production of Poly(3HB ) by Recombinant Escherichia coli Since the first demonstration that a large amount of poly(3HB ) could be synthesized in recombinant E. coli harboring the PHA biosynthesis genes of R. eutropha (Schubert et al., 1988), recombinant E. coli has been intensively examined for the production of poly(3HB ). Recombinant E. coli has been considered to be a good candidate as a producer for SCL-PHAs as it has several advantages over wild-type PHA producers such as R. eutropha and A. latus (Lee, 1997). Various metabolically engineered E. coli strains have been developed for the efficient production of poly(3HB ), with high productivity of up to 4.63 g L 1 h 1 ( Wang and Lee, 1997, 1998; Choi et al., 1998; Ahn et al., 2000). Recombinant E. coli equipped with a heterologous PHA biosynthetic pathway produces PHA in a growth-associated manner. Since poly(3HB ) is a new intracellular metabolite of E. coli, the effect of poly(3HB ) biosynthesis on the alteration of metabolic flux distribution should be evaluated in order to understand the physiological consequences of poly(3HB ) accumulation in the cells. The dissolved oxygen concentration (DOC ) is one of the important factors in poly(3HB ) production by recombinant E. coli, since oxygen often becomes limited during high cell density cultivation ( Wang and Lee, 1997; Wong et al., 1999). It was reported recently that poly(3HB ) production by recombinant E. coli could be enhanced under oxygen-limiting conditions ( Wang and Lee, 1997). To understand this phenomenon, the effects of DOC on metabolic flux distribution were examined using the MFA technique. The result of simulation using this approach suggested that 100 mol and 67 mol of poly(3HB ) were produced from 100 mol of glucose under oxygen-sufficient and oxygen-limiting conditions, respectively. It was also found that the flux through pyruvate formate-lyase increased without any change in pyruvate dehydrogenase flux under oxygen-limiting conditions, causing the accumulation of intracellular acetyl-CoA from 350 mg g 1 RCM to 600 mg g 1 RCM (van Wegen et al., 2001). This accumulated acetyl-CoA was efficiently channeled to the poly(3HB ) synthetic pathway, and more poly(3HB ) could be accumulated under the oxygen-limited condition. The amount of acetyl-CoA and NADPH available have also been found to be important factors in poly(3HB ) production by recombinant E. coli, and this agrees well with the results of MFA that acetyl-CoA and NADPH play important roles in poly(3HB ) biosynthesis by R. eutropha (Lee et al., 1996). The regulatory effects of NADPH and enzyme activities on poly(3HB ) biosynthesis were examined in recombinant E. coli XL1- 255 256 8 Metabolic Flux Analysis on the Production of Poly(3-hydroxybutyrate) Blue harboring the R. eutropha PHA biosynthesis genes. Cells were grown in various culture media including complex LuriaBertani (LB ) medium, LB supplemented with glucose, and chemically defined medium. Among these, poly(3HB ) was most favorably accumulated in LB glucose medium, which supported the highest NADPH/NADH ratio. The activity of citrate synthase, which competes with b-ketothiolase for acetyl-CoA, was also much lower when cells were cultured in LB glucose medium (Lee S. Y. et al., 1995; Lee I. Y. et al., r rmax availability of two substrates for poly(3HB ) synthesis, NADPH and acetyl-CoA. MCA was also carried out in order to evaluate thoroughly the effects of NADPH and acetyl-CoA on poly(3HB ) biosynthesis (van Wegen et al., 2001). The kinetics data of related enzymes are required for the calculation of flux control and elasticity coefficients. The b-ketothiolase catalyzes the conversion of acetyl-CoA to acetoacetyl-CoA, and it follows a ping-pong, Bi-Bi mechanism (Leaf and Srienc, 1998). The rate equation is: AB PQ Keq Kb AKa BABKq V1 P V2 Keq Kp V1 Q V2 Keq V1 PQ V2 Keq Kq V1 AP V2 Kia Keq Ka BQ Kiq 1996) also indicated the importance of the availability of NADPH on PHA biosynthesis in recombinant E. coli. Supplementation of complex nitrogen sources, oleic acid, or amino acids to the chemically defined medium significantly enhanced poly(3HB ) production. Because the biosynthesis of amino acids and oleic acid requires large amounts of reducing equivalents, poly(3HB ) production by recombinant E. coli in chemically defined media was inefficient compared with poly(3HB ) production in complex media (Lee et al., 1995). These experimental findings were supported by studies on the effects of acetyl-CoA and NADPH on the intracellular metabolic flux distribution of recombinant E. coli (Shi et al., 1999). The results of MFA suggested that in order to achieve the maximum poly(3HB ) yield, about one-half of the carbon flux should be directed to the pentose phosphate (PP ) pathway, and flux to the TCA cycle should be shut down. These two pathways affect the where r forward reaction rate (mM substrate min 1) rmax maximum forward reaction rate (mM substrate min 1) A first substrate (acetyl-CoA ) concentration (mM ) B second substrate (also acetyl-CoA ) concentration (mM ) P first product (CoA ) concentration (mM ) Q second product (acetoacetyl-CoA ) concentration (mM ) V1/V2 ratio of maximum forward reaction rate to maximum reverse reaction rate Keq equilibrium constant Ka, Kb, Kp, Kq, Kia, Kib, Kip, Kiq, various kinetic constants (mM ) The kinetics of acetoacetyl-CoA reductase has been studied to a lesser extent, but most likely follows a rapid equilibrium, random, Bi-Bi mechanism. The rate equation is: AB PQ Keq r rmax Kia Kb Kb A Ka B AB Kq V1 P V2 Keq Kp V1 Q V2 Keq V1 PQ V2 Keq 4 Production of Poly(3HB) by Recombinant Escherichia coli where A B P Q first substrate (acetoacetyl-CoA ) concentration (mM ) second substrate (NADPH ) concentration (mM ) first product ((R )-3-hydroxybutyrylCoA ) concentration (mM ) second product (NADP ) concentration (mM ) The last enzyme, PHA synthase, catalyzes the polymerization of (R )-3-hydroxybutyrylCoA, and the reaction is considered to be diffusion-limited. r R-3-hydroxybutyryl-CoA rmax Km R-3-3-hydroxybutyryl-CoA The kinetic constants for three enzymes are summarized in Table 1 (Leaf and Srienc, 1998). The results of MCA suggested that the poly(3HB ) biosynthesis flux was highly sensitive to the acetyl-CoA/CoA ratio (response coefficient 0.8) and total acetyl-CoA CoA concentration (response coefficient 0.7), while it is less sensitive to the NADPH/ NADP ratio (response coefficient 0.25) (TaTab. 1 ble 2). This means that to increase the flux to the same extent, the NADPH/NADP ratio needs to be increased more than the acetylCoA/CoA ratio. Finally, it was proposed that the overexpression of acetoacetyl-CoA reductase seems to be the most efficient way to enhance poly(3HB ) productivity, as the flux control coefficients were 0.6, 0.25, and 0.15 for acetoacetyl-CoA reductase, PHA synthase, and b-ketothiolase, respectively (see Table 2). From the results of MFA and MCA, it was found that acetyl-CoA and NADPH are the most important factors affecting poly(3HB ) production. To identify whether these two actually affect poly(3HB ) biosynthesis, two different mutant E. coli strains were examined as host strains for the production of poly(3HB ). The first strain was TA3516 harboring pJM9131 and containing the R. eutropha phb operon. In this strain, acetylCoA was expected to be overproduced because of the inactivation of phosphotransacetylase and acetate kinase (Shi et al., 1999). The production rate of lactic acid decreased significantly, whilst those of pyruvate and acetic acid decreased only slightly. However, Kinetic constants for b-ketothiolase, acetoacetyl-CoA reductase and PHA synthasea b-Ketothiolase Keq V1/V2 Ka Kb Kp 4 î 10 5 2.5 î 10 4 3.78 î 10 3 mM 840 mM 31.4 mM Kq Kia Kib Kip Kiq 64.6 mM 5.96 mM 841 mM 12.4 mM 1.62 î 10 2 mM 500 5 mM 19 mM Kp, Kip Kq, Kiq 16.5 mM 31 mM Acetoacetyl-CoA reductase Keq Ka, Kia Kb, Kib PHA synthase Km a 720 mM Data taken from Leaf and Srienc (1998). 257