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Metabolic Engineering of Escherichia Coli for the Production of Polyhydroxyalkanoates
Copyright © IFAC Computer Applications in Biotechnology, Osaka, Japan, 1998
MET ABOLlC ENGINEERING OF ESCHERICHIA COLI FOR THE PRODUCTION OF POLYHYDROXYALKANOATES
Sang Yup Lee and Jong-il Choi
Department o/Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute o[Science and Technology, 373-1 Kusong-dong, Yusong-gu, Tae/on 305- 701, Korea. e-mail : leesy@ sorak.kaist.ac.kr
Abstract: The current knowledge on the development of re comb in ant Escherichia coli for the production of polyhydroxyalkanoates (PHAs) is provided. Recombinant E. coli transformed with a stable high copy number plasmid containing the Ralstonia eutropha PHA biosynthesis genes accumulated large amount of poly(3-hydroxybutyrate) [P(3HB)] , and P(3HB) production was enhanced by suppressing cell filamentation during P(3HB) accumulation . Other PH As were synthesized by recombinant E. coli. Several different recombinant E. coli strains were developed to produce PHAs from cheap substrates. The molecular weight of P(3HB) was controlled in recombinant E. coli. Copyright © 1998lFAC Keywords : Biotechnology, Fermentation processes
I. INTRODUCTION
chain-length PHAs (MCL-PHA) consisting of 6 - 14 carbon atoms (Steinbuchel, 1991). One of the major problems of employing PHAs in a wide range of applications is the high production cost of PHAs compared with petrochemical-based polymers (Lee, 1996a). Therefore, much effort has been devoted to reduce the production cost of PHA by the development of better bacterial strains and more efficient fermentation and recovery processes.
Plastic materials have become indispensable in our modern life. Recently, problems caused by the accumulation of non-degradable plastic materials on our environment have created much interest in the development of biodegradable polymers. Among the several biodegradable polymers under development, polyhydroxyalkanoates (PH As) have attracted much attention because of their similar material properties to conventional petrochemical-derived plastics and elastomers, and complete biodegradability under various environments (Anderson et aI. , 1990; Doi, 1990; Lee , 1996a; Steinbuchel, 1991). PHAs are the polymers of various hydroxyalkanoates, which are accumulated as a carbon and energy storage material in various microorganisms usually under the condition of limiting nutritional elements in the presence of excess carbon source (Anderson et aI., 1990; Lee, I 996a). PH As can be divided into two groups depending on the number of carbon atoms of the monomeric units : short-chain-Iength PHAs (SCLPHA) consisting of 3 - 5 carbon atoms and medium-
Metabolic engineering approaches can be used to modify or introduce new metabolic pathways to enhance PHA production capacity, to broaden the utilizable substrate range, and to produce novel polymers. With the recent advances in our understanding of the biochemistry of PHA biosynthesis and cloning of the PHA biosynthesis genes from a number of different bacteria (Lee, I 996b; Steinbuchel et aI., 1995), it is possible to metabolically engineer various organisms for the production of PHAs. In this paper, development and use of metabolically engineered Escherichia coli strains for the production of PHAs are reviewed .
337
The P(3HB) concentration of higher than 80 g/L with the productivity of greater than 2 g P(3HB)/L-h could be obtained by the pH-stat fed-batch culture of recombinant E. coli in a complex medium containing glucose (Kim et aI. , 1992 ; Lee et aI., 1994c). In a defined medium , however, only less than 25 g/ L of P(3HB) could be produced by the similar fed-batch cultures (Lee et af. , I 994a). In a defined medium , however, P(3HB) production could significantly be enhanced by the addition of small amounts of various complex nitrogen sources (Lee and Chang, 1994). amino acids (Lee et aI., 1995), or oleic acid (Lee et aI. , 1995). It was later found that the enhancing effect observed in these conditions was due to the availability of more acetyl-CoA and/or NADPH , a cofactor of the reductase in the PHA biosynthetic pathway for PHA biosynthesis (Lee et aI., 1996). Recombinant E. coli accumulated a large amount of P(3HB) when the NADPH level and NADPHINADP ratio were high . The activity of citrate synthase, which competes with ~-ketothiolase for acetyl-CoA. was much lower and more acetyl-CoA were available for P(3HB) biosynthetic pathway at high level ofNADPH and NADPHINADP ratio .
2. STRAIN DEVELOPMENT
Ralstonia eutropha (fonnerly Alcaligenes eutrophus) is the bacterium that has most often been employed for the production of SCL-PHAs since it accumulates a large amount of polymer in a relatively simple medium (Doi, 1990; Lee, 1996b). In R. eutropha, acetyl-CoA is converted to poly(3-hydroxybutyrate) [P(3HB)] in three enzymatic steps (Anderson et 01., 1990; Steinbuchel, 1991). A biosynthetic ~ ketothiolase catalyses the fonnation of a carboncarbon bond by biological Claisen condensation of two acetyl-CoA moieties . An NADPH-dependent acetoacetyl-CoA reductase stereoselectively reduces acetoacetyl-CoA to 0-( - )-3-hydroxybutyryl-CoA, which is then linked to the growing chain of P(3HB) by the PHA synthase. The PHA biosynthesis genes of R. elltropha were cloned in E. coli, and were sequenced and characterized in detail (Peoples and Sinskey, 1989; Schubert et aI. , 1988 ; Slater et 01., 1988). The three genes fonn an operon in the order of phaC-A-B, coding for PHA synthase, ~ - ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase , respectively . During the cloning studies, it was shown that these three genes were constitutively expressed from the native promoter in E. coli.
4. CELLULAR ENGINEERING FOR FILAMENTA TION SUPPRESSION It was interesting to observe that cells of recombinant E. coli underwent considerable filamentation during synthesis and accumulation of P(3HB) (Lee, 1994b; Lee et 01. , 1994b). It was thought that P(3HB) production might be enhanced by suppressing cell filamentation. Filamentation of cells could be suppressed by overexpressing an essential cell division protein FtsZ (Lee, 1994a; Lee , 1994b). This filamentation-suppressed recombinant E. coli strain allowed production of P(3HB) to a high concentration of 104 g/L with P(3HB) content of 70% and productivity of 2 g P(3HB)/L-h in a defined medium (Wang and Lee , 1998). Further optimization of growth medium and culture condition resulted in P(3HB) concentration, P(3HB) content, and productivity of 149 g/L, 77%, and 3.4 g P(3HB)/ L-h, respectively , in a defined medium (Wang and Lee , 1997). These values are much higher than those obtainable with recombinant E. coli strains without filamentation-suppression in a defined medium (Lee et af., 1994a). These results clearly demonstrated that cellular properties that do not have direct relation to the PHA biosynthetic pathway can have a significant effect on PHA production.
Several plasmids possessing different copy numbers (medium and high copy numbers) containing the R. eutropha PHA biosynthesis genes were constructed, and subsequently compared in E. coli for their effectiveness in PHA production (Lee et 01. , 1994c). It was found from flask and fed-batch cultures that the use of a stable high copy number plasmid was necessary for the efficient biosynthesis of P(3HB) in recombinant E. coli. Comparison of more than 30 different E. coli strains transfonned with a stable high copy number plasmid containing the R. eutropha PHA biosynthesis genes showed that there was a large variation in the ability of synthesizing P(3HB) among the different E. coli strains (Lee and Chang, 1995c ; Lee et 01., I 994b ). Two of these recombinant strains, XL I-Blue and B harboring a stable high copy number plasmid. synthesized P(3HB) at the highest rate to the highest content, and therefore, were employed in further studies.
3. PRODUCTION OF P(3HB) It was found that recombinant E. coli does not require the limitation of a specific nutritional element for the production of P(3HB), which is different from the most of the natural PHA producers (Lee et aI. , 1994a). Various fed-batch culture techniques were examined , and the pH-stat feeding strategy was found to be the best for the PHA production by recombinant E. coli.
5. PRODUCTION OF POL Y(3HYDROXYBUTYRA TE-CO-3HYDROXYV ALERA TE)
338
For the synthesis of poly(3-hydroxybutyrate-co-3hydroxyvalerate) [P(3HB-co-3HV)] copolymer, 3hydroxyvaleryl-CoA should be generated in the cell. The 3-hydroxyvaleryl-CoA moiety can usually be generated by co-feeding short-chain-Iength odd carbon numbered fatty acids (e.g., propionate and valerate) together with a major carbon source. However, utilization of these acids is rather inefficient in E. coli. Therefore, a mutant E. coli strain LS5218 (jadR atoC) was employed (Slater et aI. , 1992; Yim et aI. , 1995). The mutations carried by this strain allow constitutive expression of the enzymes involved in the transport and utilization of fatty acids . The copolymer P(3HB-co-3HV) could be synthesized by a recombinant E. coli strain LS5218 harboring the R. eutropha PHA biosynthesis genes when propionate or valerate was added as a co-substrate. The P(3HB-co3HV) copolymer consisting of up to 40 mol% of 3HV could be produced. An alternative method that allowed synthesis of P(3HB-co-3HV) using propionate or valerate as a cosubstrate in non-fadR atoC E. coli strains was also investigated (Yim et aI. , 1996). By induction with acetate and/or oleate, nonfadR atoC strains of E. coli could efficiently produce P(3HB-co-3HV) from propionate. This is an important strategy for the production of P(3HB-co3HV) since one does not have to rely on fadR atoC mutant strain, which usually does not grow well to a high density . Another approach taken was production of P(3HB-co-3HV) by supplementing a small amount of valine (Eschenlauer et aI., 1996). Valine stimulates threonine dehydrogenase and inhibits acetolactate synthase to accumulate a-ketobutyrate . In turn , aketobutyrate is converted to propionyl-CoA by the pyruvate dehydrogenase complex.
cultivated in a medium contammg 0.4% 4HB and 0.5% glucose, P(4HB) was produced. In an independent study, poly(3HB-co-4HB) was produced in recombinant E. coli harboring the R. eutropha PHA biosynthetic genes and the C. kluyveri succinate degradation genes (Valentin and Dennis, 1998). Three enzymes, 4-hydroxybutyrate dehydrogenase, succinic semialdehyde dehydrogenase, and 4-hydroxybutyrylCoA :CoA transferase of C. kluyveri together with the R. eutropha PHA biosynthesis genes were cloned and expressed in E. coli. Poly(3HB-co-4HB) containing 2.8 mol% 4HB could be accumulated up to 50% of dry cell weight in complex medium containing glucose . These studies demonstrated that introduction of metabolic pathways that generate novel precursors of PHAs allows production of the corresponding novel PHAs.
7. BROADENING UTILIZABLE SUBSTRA TE RANGE Since the cost of carbon substrate has significant effect on the total production cost of PHAs, agricultural waste products such as cheese whey , molasses, and hemicellulose hydrolysates would be economically attractive carbon sources if they can be utilized by the PHA producers (Choi and Lee, 1997; Lee and Chang, 1995b). To produce PHAs from these inexpensive carbon substrates, several recombinant E. coli strains have been developed . To make recombinant E. coli utilize sucrose, the Bacillus subtilis levanase gene was transferred into XL I-Blue and B strains harboring the plasmid containing the R. eutropha PHA biosynthesis genes . However, these recombinant E. coli strains did not grow on sucrose (S.Y. Lee, unpublished results). Several non-K 12 E. coli strains including W (Lee and Chang, 1993 ; Lee and Chang, 1995a) and JMU213 (Zhang et aI. , 1994) were found to be able to utilize sucrose. The PHA biosynthesis genes were transferred into these strains, and P(3HB) could be produced from sucrose (and thus from cane and beet molasses) . From this experience, it was concluded that it might be better to employ an E. coli strain that can utilize an inexpensive carbon substrate to start with, instead of making it utilize this substrate by introducing the substrate utilization genes. This approach has been successfully employed to develop recombinant E. coli strains that can efficiently produce PHAs from lactose (Lee et aI. , 1997) and xylose (Lee, 1998), and therefore, cheese whey and hemicellulose hydrolysate.
6. PRODUCTION OF OTHER PHAS Production of MCL-PHA in recombinant E. coli has recently been reported (Langenbach et aI., 1997). The Pseudomonas aeruginosa PHA synthase gene phaC 1 under the control of the E. coli lac promoter was transferred into E. coli. When induced with IPTG , recombinant E. coli harboring the P. aerllginosa phaC 1 accumulated MCL-PHA from decanoate . It was suggested that intermediates of the fatty acid poxidation pathway were directed to MCL-PHA biosynthesis in E. coli. Recombinant E. coli LS 1298, a fatty acid degradation mutant strain , accumulated MCL-PHA to 21 % of DCW, when cultivated in LB medium containing 0.5% (w/v) sodium decanoate . Production of poly(4-hydroxybutyrate) [P(4HB)] in recombinant E. coli has also been reported (Steinbuchel, presentation at the E-MRS ' 97). When recombinant E. coli harboring the Clostridium kluyveri 4-hydroxybutyl-CoA dehydrogenase gene as well as the R. elltropha PHA synthase gene was
8. CONTROL OF MOLECULAR WEIGHT Molecular weight of polymer is one of the important parameters that determines the material properties of the polymer. It has recently been reported that the
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molecular weight of P(3HB) could be controlled by modulating the activity of the PHA synthase in recombinant E. coli harboring the R. eutropha PHA biosynthesis genes (Sim et aI., 1997). The activity of the PHA synthase was controlled by employing different plasmid constructs with either weak constitutive promoter or IPTG inducible promoter. The molecular weight of P(3HB) decreased with increasing PHA synthase activity. An extremely high molecular weight (4 x IO J kDa) of P(3HB) could be obtained with a recombinant E. coli expressing the PHA synthase at low level (by constitutive expression from the R. eutropha promoter). The polydispersity index could also be controlled by modulating the PHA synthase activity. Because the molecular weight and polydispersity index affect material properties and processibility of polymer, PHAs having different, but well-defined characteristics can be produced by this strategy. However, it is not desirable to use an expensive inducer (e. g., IPTG) to control the PHA synthase activity because it will increase the PHA production cost significantly. Therefore, it is interesting to find that the molecular weight of P(3HB) can be also controlled in recombinant E. coli constitutively expressing the PHA biosynthetic enzymes by varying the culture pH (Kusaka et al.. 1997).
9. CONCLUSION Metabolic engineering strategies have been employed to enhance PHA biosynthetic capacity, to broaden the utilizable substrate range and to produce novel PHAs. Several advantages were found by employing metabolically engineered E. coli for the production of PHAs (Lee, 1997; Lee and Chang, 1996): (i) fast growth to a high cell density, (ii) accumulation of a large amount of polymers, (iii) ability to utilize several inexpensive carbon sources, (iv) fragility of cells accumulating PH As allowing relatively easy purification of PHAs (Lee, 1996c; Middelberg et aI. , 1995), and (v) the lack of intracellular depolymerases which degrade the accumulated PHAs. These advantages together with the cultivation strategies developed to achieve PHA productivity higher than 3.4 g PHA/L-h allow recombinant E. coli to become a strong candidate as a PHA producer.
ACKNOWLEDGEMENTS The research of the author on PHAs has been supported by grants provided by the KAIST, the KOSEF, and MOST, and by LG Chemical Ltd .
REFERENCES
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Anderson, A.J. and E.A. Dawes (1990). Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev., 54,450-472. Choi , 1. and S.Y. Lee (1997). Process analysis and economic evaluation for poly(3-hydroxybutyrate} production by fermentation. Bioprocess. Eng., 17, 335-342 . Doi, Y. (1990). Microbial polyesters . VCH, New York. Eschenlauer, A,C. , S.K . Stoup, F. Srienc and D.A. Somers (1996) . Production of heteropolymeric polyhydroxyalkanoate in Escherichia coli from a single carbon source . 1nl. 1. BioI. Macromol., 19, 121-130. Kim B.S. , S.Y . Lee and H.N. Chang (1992). Production of poly-~-hydroxybutyrate by fed-batch culture of recombinant Escherichia coli. Biotechnol. Left. , 14, 811-816. Kusaka S., H. Abe, S.Y . Lee and Y. Doi (I 997}. Molecular mass of poly[(R}-3-hydroxybutyric acid] produced in a recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 47, 140-143 . Langenbach , S., B.H .A. Rehm and A. Steinbuchel (1997). Functional expression of the PHA synthase gene phaC I from Pseudomonas aeruginosa in Escherichia coli results in poly(3hydroxyalkanoate} synthesis. FEMS Microbiol. Left., 150, 303-309. Lee, I.Y. , M.K . Kim, Y.H . Park and S.Y. Lee (1996). Regulatory effects of cellular nicotinamide nucleotide and enzyme activities on poly(3hydroxybutyrate} synthesis in recombinant Escherichia coli. Biotechnol. Bioeng. , 52, 707-712 . Lee, S.Y. (1994a). Effects of growth temperature and nutritional components on the synthesis of poly(3hydroxybutyrate} by filamentation-suppressed recombinant Escherichia coli. Kor. 1. Appl. Microbiol. Biotechnol. , 22, 614-620. Lee, S.Y. (1994b) . Suppression of filamentation in recombinant Escherichia coli by amplified FtsZ activity . Biotechnol. Lelt., 16. 1247-1252. Lee, S. Y. (l996a). Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng., 49,1-14. Lee, S. Y. (1996b). Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria. Trends Biotechnol. , 14, 431- 438 . Lee, S.Y. (1996c) . Poly(3-hydroxybutyrate} extrusion by cells of recombinant Escherichia coli. 1. Microbiol. Biotechnol. , 6, 147-149. Lee, S.Y. (l997) . E. coli moves into the plastic age . Nature Biotechnol., IS, 17-18. Lee , S.Y. (I 998}. Production of poly(3hydroxybutyrate} from xylose by recombinant Escherichia coli. Bioprocess Eng in press. Lee, S.Y. and H.N. Chang (1993). High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnol. Left., IS, 971-974 .
Sim, S.J ., K.D. Snell, S.A. Hogan, J. Stubbe, C. Rha and A.J . Sinskey (1997). PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nature Biotechnol. , 15, 63-67. Slater, S.c., W.H. Voige and D.E. Dennis (1988). Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H 16 poly-P-hydroxybutyrate biosynthetic pathway. J Bacteriol. , 170, 44314436. Slater, S., T. Gallaher and D. Dennis (1992). Production of poly-(3-hydroxybutyrate-co-3hydroxyvalerate) in a recombinant Escherichia coli strain. Appl. Environ. Microbiol. , 58, 1089-1094. Steinbuchel, A. (199 I). Polyhydroxyalkanoic acids. In : Biomaterials: novel materials from biological sources (D. Byrom, Ed.), pp. 124-213 . Stockton, New York. Steinbuchel , A, K. Aerts, W. Babel, C. Follner, M. Liebergesell, M.H . Madkour, F. Mayer, U.P. Furst, A. Pries, H.E. Valentin and R. Wieczorek (1995). Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusion. Can. J Microbiol. , 41(Suppl. 1), 94-105 . Valentin, H.E. and D. Dennis (1998). Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose. J Biotechnol. in press. Wang, F. and S.Y. Lee (1997). Production ofpoly(3hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli. Appl. Environ. Microbiol. , 63, 4765-4769. Wang, F. and S.Y. Lee (1998). High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium . Biotechnol. Bioeng., in press. Yim , K.S., S.Y. Lee and H.N. Chang (1995). Effect of acetic acid and metabolic intermediates on poly(3hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant Escherichia coli. Kor. J Chem. Eng, 12,264-268. Yim , K.S., S.Y. Lee and H.N. Chang (1996). Synthesis of poly(3-hydroxybutyrate-co-3hydroxyvalerate) by recombinant Escherichia coli. Biotechnol. Bioeng, 49,495-503 . Zhang, H. , V. Obias, K. Gonyer and D. Dennis ( 1994). Production of polyhydroxyalkanoates in sucrose-utilizing recombinant Escherichia coli and Klebsiella strains. Appl. Environ. Microbiol. , 60, 1198-\205 .
Lee S.Y . and H.N. Chang (1994). Effect of complex nitrogen source on the synthesis and accumulation of poly(3-hydroxybutyric acid) by recombinant Escherichia coli in flask and fed-batch cultures. J Environ. Polymer Degrad., 2, 169-176. Lee, S.Y. and H.N. Chang (1995a). Effect of complex nitrogen sources on poly(3-hydroxybutyric acid) synthesis by recombinant Escherichia coli using sucrose as a carbon source. Kor. J Biotechnol. Bioeng , 10, 137-142. Lee, S.Y. and H.N . Chang (1995b). Production of poly-(hydroxyalkanoic acid) . Adv. Biochem. Eng. Biotechnol , 52, 27-58 Lee S.Y. and H.N. Chang (1995c). Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studied. Can. J Microhiol. , 41(Suppl. 1),207-215 . Lee, S.Y. and H.N. Chang (1996). Characteristics of poly(3-hydroxybutyric acid) synthesis by recombinant Escherichia coli. Ann. NY Acad. Sci., 782, 133-142. Lee S.Y. , H.N. Chang and Y.K. Chang (1994a). Production of polyW-hydroxybutyric acid) by recombinant Escherichia coli. Ann. NY Acad. Sci., 721,43-53. Lee S.Y., K.M . Lee, H.N . Chang and A. Steinbuchel (I 994b). Comparison of Eschericia coli strains for of poly-(3synthesis and accumulation hydroxybutyric acid), and morphological changes . Biotechnol. Bioeng , 44, 1337- I 347. Lee, S.Y ., K.S . Yim, H.N. Chang and Y.K . Chang ( I 994c). Construction of plasm ids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) in Escherichia coli. J Biotechnol. , 32, 203-2 I I. Lee S.Y., Y.K. Lee and H.N. Chang (1995). Stimulatory effects of amino acids and oleic acid on poly(3-hydroxybutyric acid) synthesis by recombinant Escherichia coli. J Ferment. Bioeng, 79, 177- I 80. Lee, S.Y., A.P.J. Middelberg and Y.K. Lee (1997). Production of poly(3-hydroxybutyrate) from whey using recombinant Escherichia coli. Biotechnol. Left., 19, 1033- 1035. Middelberg, A.P.J., S.Y. Lee, J. Martin , D.R.G. Williams and H.N. Chang (1995). Size analysis of poly(3-hydroxybutyric acid) granules produced in recombinant Escherichia coli. Biotechnol. Left. , 17, 205-2 I O. Peoples, O.P. and A.J. Sinskey (1989). Poly-phydroxybutyrate biosynthesis in Alcaligenes eutrophus HI 6. Identification and characterization of the PHB polymerase gene (phhC) . J BioI. Chem. , 264, 15298-15303 . Schubert, P, A. Steinbuchel and H.G. Schlegel (1988). Cloning of the Alcaligenes eutrophus poly-phydroxybutyrate synthetic pathway and synthesis of PHB in Escherichia coli. J Bacterio/ .. 170, 58375847 .
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MET ABOLlC ENGINEERING OF ESCHERICHIA COLI FOR THE PRODUCTION OF POLYHYDROXYALKANOATES
Sang Yup Lee and Jong-il Choi
Department o/Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute o[Science and Technology, 373-1 Kusong-dong, Yusong-gu, Tae/on 305- 701, Korea. e-mail : leesy@ sorak.kaist.ac.kr
Abstract: The current knowledge on the development of re comb in ant Escherichia coli for the production of polyhydroxyalkanoates (PHAs) is provided. Recombinant E. coli transformed with a stable high copy number plasmid containing the Ralstonia eutropha PHA biosynthesis genes accumulated large amount of poly(3-hydroxybutyrate) [P(3HB)] , and P(3HB) production was enhanced by suppressing cell filamentation during P(3HB) accumulation . Other PH As were synthesized by recombinant E. coli. Several different recombinant E. coli strains were developed to produce PHAs from cheap substrates. The molecular weight of P(3HB) was controlled in recombinant E. coli. Copyright © 1998lFAC Keywords : Biotechnology, Fermentation processes
I. INTRODUCTION
chain-length PHAs (MCL-PHA) consisting of 6 - 14 carbon atoms (Steinbuchel, 1991). One of the major problems of employing PHAs in a wide range of applications is the high production cost of PHAs compared with petrochemical-based polymers (Lee, 1996a). Therefore, much effort has been devoted to reduce the production cost of PHA by the development of better bacterial strains and more efficient fermentation and recovery processes.
Plastic materials have become indispensable in our modern life. Recently, problems caused by the accumulation of non-degradable plastic materials on our environment have created much interest in the development of biodegradable polymers. Among the several biodegradable polymers under development, polyhydroxyalkanoates (PH As) have attracted much attention because of their similar material properties to conventional petrochemical-derived plastics and elastomers, and complete biodegradability under various environments (Anderson et aI. , 1990; Doi, 1990; Lee , 1996a; Steinbuchel, 1991). PHAs are the polymers of various hydroxyalkanoates, which are accumulated as a carbon and energy storage material in various microorganisms usually under the condition of limiting nutritional elements in the presence of excess carbon source (Anderson et aI., 1990; Lee, I 996a). PH As can be divided into two groups depending on the number of carbon atoms of the monomeric units : short-chain-Iength PHAs (SCLPHA) consisting of 3 - 5 carbon atoms and medium-
Metabolic engineering approaches can be used to modify or introduce new metabolic pathways to enhance PHA production capacity, to broaden the utilizable substrate range, and to produce novel polymers. With the recent advances in our understanding of the biochemistry of PHA biosynthesis and cloning of the PHA biosynthesis genes from a number of different bacteria (Lee, I 996b; Steinbuchel et aI., 1995), it is possible to metabolically engineer various organisms for the production of PHAs. In this paper, development and use of metabolically engineered Escherichia coli strains for the production of PHAs are reviewed .
337
The P(3HB) concentration of higher than 80 g/L with the productivity of greater than 2 g P(3HB)/L-h could be obtained by the pH-stat fed-batch culture of recombinant E. coli in a complex medium containing glucose (Kim et aI. , 1992 ; Lee et aI., 1994c). In a defined medium , however, only less than 25 g/ L of P(3HB) could be produced by the similar fed-batch cultures (Lee et af. , I 994a). In a defined medium , however, P(3HB) production could significantly be enhanced by the addition of small amounts of various complex nitrogen sources (Lee and Chang, 1994). amino acids (Lee et aI., 1995), or oleic acid (Lee et aI. , 1995). It was later found that the enhancing effect observed in these conditions was due to the availability of more acetyl-CoA and/or NADPH , a cofactor of the reductase in the PHA biosynthetic pathway for PHA biosynthesis (Lee et aI., 1996). Recombinant E. coli accumulated a large amount of P(3HB) when the NADPH level and NADPHINADP ratio were high . The activity of citrate synthase, which competes with ~-ketothiolase for acetyl-CoA. was much lower and more acetyl-CoA were available for P(3HB) biosynthetic pathway at high level ofNADPH and NADPHINADP ratio .
2. STRAIN DEVELOPMENT
Ralstonia eutropha (fonnerly Alcaligenes eutrophus) is the bacterium that has most often been employed for the production of SCL-PHAs since it accumulates a large amount of polymer in a relatively simple medium (Doi, 1990; Lee, 1996b). In R. eutropha, acetyl-CoA is converted to poly(3-hydroxybutyrate) [P(3HB)] in three enzymatic steps (Anderson et 01., 1990; Steinbuchel, 1991). A biosynthetic ~ ketothiolase catalyses the fonnation of a carboncarbon bond by biological Claisen condensation of two acetyl-CoA moieties . An NADPH-dependent acetoacetyl-CoA reductase stereoselectively reduces acetoacetyl-CoA to 0-( - )-3-hydroxybutyryl-CoA, which is then linked to the growing chain of P(3HB) by the PHA synthase. The PHA biosynthesis genes of R. elltropha were cloned in E. coli, and were sequenced and characterized in detail (Peoples and Sinskey, 1989; Schubert et aI. , 1988 ; Slater et 01., 1988). The three genes fonn an operon in the order of phaC-A-B, coding for PHA synthase, ~ - ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase , respectively . During the cloning studies, it was shown that these three genes were constitutively expressed from the native promoter in E. coli.
4. CELLULAR ENGINEERING FOR FILAMENTA TION SUPPRESSION It was interesting to observe that cells of recombinant E. coli underwent considerable filamentation during synthesis and accumulation of P(3HB) (Lee, 1994b; Lee et 01. , 1994b). It was thought that P(3HB) production might be enhanced by suppressing cell filamentation. Filamentation of cells could be suppressed by overexpressing an essential cell division protein FtsZ (Lee, 1994a; Lee , 1994b). This filamentation-suppressed recombinant E. coli strain allowed production of P(3HB) to a high concentration of 104 g/L with P(3HB) content of 70% and productivity of 2 g P(3HB)/L-h in a defined medium (Wang and Lee , 1998). Further optimization of growth medium and culture condition resulted in P(3HB) concentration, P(3HB) content, and productivity of 149 g/L, 77%, and 3.4 g P(3HB)/ L-h, respectively , in a defined medium (Wang and Lee , 1997). These values are much higher than those obtainable with recombinant E. coli strains without filamentation-suppression in a defined medium (Lee et af., 1994a). These results clearly demonstrated that cellular properties that do not have direct relation to the PHA biosynthetic pathway can have a significant effect on PHA production.
Several plasmids possessing different copy numbers (medium and high copy numbers) containing the R. eutropha PHA biosynthesis genes were constructed, and subsequently compared in E. coli for their effectiveness in PHA production (Lee et 01. , 1994c). It was found from flask and fed-batch cultures that the use of a stable high copy number plasmid was necessary for the efficient biosynthesis of P(3HB) in recombinant E. coli. Comparison of more than 30 different E. coli strains transfonned with a stable high copy number plasmid containing the R. eutropha PHA biosynthesis genes showed that there was a large variation in the ability of synthesizing P(3HB) among the different E. coli strains (Lee and Chang, 1995c ; Lee et 01., I 994b ). Two of these recombinant strains, XL I-Blue and B harboring a stable high copy number plasmid. synthesized P(3HB) at the highest rate to the highest content, and therefore, were employed in further studies.
3. PRODUCTION OF P(3HB) It was found that recombinant E. coli does not require the limitation of a specific nutritional element for the production of P(3HB), which is different from the most of the natural PHA producers (Lee et aI. , 1994a). Various fed-batch culture techniques were examined , and the pH-stat feeding strategy was found to be the best for the PHA production by recombinant E. coli.
5. PRODUCTION OF POL Y(3HYDROXYBUTYRA TE-CO-3HYDROXYV ALERA TE)
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For the synthesis of poly(3-hydroxybutyrate-co-3hydroxyvalerate) [P(3HB-co-3HV)] copolymer, 3hydroxyvaleryl-CoA should be generated in the cell. The 3-hydroxyvaleryl-CoA moiety can usually be generated by co-feeding short-chain-Iength odd carbon numbered fatty acids (e.g., propionate and valerate) together with a major carbon source. However, utilization of these acids is rather inefficient in E. coli. Therefore, a mutant E. coli strain LS5218 (jadR atoC) was employed (Slater et aI. , 1992; Yim et aI. , 1995). The mutations carried by this strain allow constitutive expression of the enzymes involved in the transport and utilization of fatty acids . The copolymer P(3HB-co-3HV) could be synthesized by a recombinant E. coli strain LS5218 harboring the R. eutropha PHA biosynthesis genes when propionate or valerate was added as a co-substrate. The P(3HB-co3HV) copolymer consisting of up to 40 mol% of 3HV could be produced. An alternative method that allowed synthesis of P(3HB-co-3HV) using propionate or valerate as a cosubstrate in non-fadR atoC E. coli strains was also investigated (Yim et aI. , 1996). By induction with acetate and/or oleate, nonfadR atoC strains of E. coli could efficiently produce P(3HB-co-3HV) from propionate. This is an important strategy for the production of P(3HB-co3HV) since one does not have to rely on fadR atoC mutant strain, which usually does not grow well to a high density . Another approach taken was production of P(3HB-co-3HV) by supplementing a small amount of valine (Eschenlauer et aI., 1996). Valine stimulates threonine dehydrogenase and inhibits acetolactate synthase to accumulate a-ketobutyrate . In turn , aketobutyrate is converted to propionyl-CoA by the pyruvate dehydrogenase complex.
cultivated in a medium contammg 0.4% 4HB and 0.5% glucose, P(4HB) was produced. In an independent study, poly(3HB-co-4HB) was produced in recombinant E. coli harboring the R. eutropha PHA biosynthetic genes and the C. kluyveri succinate degradation genes (Valentin and Dennis, 1998). Three enzymes, 4-hydroxybutyrate dehydrogenase, succinic semialdehyde dehydrogenase, and 4-hydroxybutyrylCoA :CoA transferase of C. kluyveri together with the R. eutropha PHA biosynthesis genes were cloned and expressed in E. coli. Poly(3HB-co-4HB) containing 2.8 mol% 4HB could be accumulated up to 50% of dry cell weight in complex medium containing glucose . These studies demonstrated that introduction of metabolic pathways that generate novel precursors of PHAs allows production of the corresponding novel PHAs.
7. BROADENING UTILIZABLE SUBSTRA TE RANGE Since the cost of carbon substrate has significant effect on the total production cost of PHAs, agricultural waste products such as cheese whey , molasses, and hemicellulose hydrolysates would be economically attractive carbon sources if they can be utilized by the PHA producers (Choi and Lee, 1997; Lee and Chang, 1995b). To produce PHAs from these inexpensive carbon substrates, several recombinant E. coli strains have been developed . To make recombinant E. coli utilize sucrose, the Bacillus subtilis levanase gene was transferred into XL I-Blue and B strains harboring the plasmid containing the R. eutropha PHA biosynthesis genes . However, these recombinant E. coli strains did not grow on sucrose (S.Y. Lee, unpublished results). Several non-K 12 E. coli strains including W (Lee and Chang, 1993 ; Lee and Chang, 1995a) and JMU213 (Zhang et aI. , 1994) were found to be able to utilize sucrose. The PHA biosynthesis genes were transferred into these strains, and P(3HB) could be produced from sucrose (and thus from cane and beet molasses) . From this experience, it was concluded that it might be better to employ an E. coli strain that can utilize an inexpensive carbon substrate to start with, instead of making it utilize this substrate by introducing the substrate utilization genes. This approach has been successfully employed to develop recombinant E. coli strains that can efficiently produce PHAs from lactose (Lee et aI. , 1997) and xylose (Lee, 1998), and therefore, cheese whey and hemicellulose hydrolysate.
6. PRODUCTION OF OTHER PHAS Production of MCL-PHA in recombinant E. coli has recently been reported (Langenbach et aI., 1997). The Pseudomonas aeruginosa PHA synthase gene phaC 1 under the control of the E. coli lac promoter was transferred into E. coli. When induced with IPTG , recombinant E. coli harboring the P. aerllginosa phaC 1 accumulated MCL-PHA from decanoate . It was suggested that intermediates of the fatty acid poxidation pathway were directed to MCL-PHA biosynthesis in E. coli. Recombinant E. coli LS 1298, a fatty acid degradation mutant strain , accumulated MCL-PHA to 21 % of DCW, when cultivated in LB medium containing 0.5% (w/v) sodium decanoate . Production of poly(4-hydroxybutyrate) [P(4HB)] in recombinant E. coli has also been reported (Steinbuchel, presentation at the E-MRS ' 97). When recombinant E. coli harboring the Clostridium kluyveri 4-hydroxybutyl-CoA dehydrogenase gene as well as the R. elltropha PHA synthase gene was
8. CONTROL OF MOLECULAR WEIGHT Molecular weight of polymer is one of the important parameters that determines the material properties of the polymer. It has recently been reported that the
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molecular weight of P(3HB) could be controlled by modulating the activity of the PHA synthase in recombinant E. coli harboring the R. eutropha PHA biosynthesis genes (Sim et aI., 1997). The activity of the PHA synthase was controlled by employing different plasmid constructs with either weak constitutive promoter or IPTG inducible promoter. The molecular weight of P(3HB) decreased with increasing PHA synthase activity. An extremely high molecular weight (4 x IO J kDa) of P(3HB) could be obtained with a recombinant E. coli expressing the PHA synthase at low level (by constitutive expression from the R. eutropha promoter). The polydispersity index could also be controlled by modulating the PHA synthase activity. Because the molecular weight and polydispersity index affect material properties and processibility of polymer, PHAs having different, but well-defined characteristics can be produced by this strategy. However, it is not desirable to use an expensive inducer (e. g., IPTG) to control the PHA synthase activity because it will increase the PHA production cost significantly. Therefore, it is interesting to find that the molecular weight of P(3HB) can be also controlled in recombinant E. coli constitutively expressing the PHA biosynthetic enzymes by varying the culture pH (Kusaka et al.. 1997).
9. CONCLUSION Metabolic engineering strategies have been employed to enhance PHA biosynthetic capacity, to broaden the utilizable substrate range and to produce novel PHAs. Several advantages were found by employing metabolically engineered E. coli for the production of PHAs (Lee, 1997; Lee and Chang, 1996): (i) fast growth to a high cell density, (ii) accumulation of a large amount of polymers, (iii) ability to utilize several inexpensive carbon sources, (iv) fragility of cells accumulating PH As allowing relatively easy purification of PHAs (Lee, 1996c; Middelberg et aI. , 1995), and (v) the lack of intracellular depolymerases which degrade the accumulated PHAs. These advantages together with the cultivation strategies developed to achieve PHA productivity higher than 3.4 g PHA/L-h allow recombinant E. coli to become a strong candidate as a PHA producer.
ACKNOWLEDGEMENTS The research of the author on PHAs has been supported by grants provided by the KAIST, the KOSEF, and MOST, and by LG Chemical Ltd .
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