- Email: [email protected]
Metabolic improvements and use of inexpensive carbon sources in microbial production of polyhydroxyalkanoates
JOURNALOF BIOSCIENCEAND BIOENGINEERING Vol. 94, No. 6,579-584.2002
REVIEW Metabolic Improvements and Use of Inexpensive Carbon Sources in Microbial Production of Polyhydroxyalkanoates TAKEHARU
TSUGE’
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan’ Received 23 July 2002IAccepted
4 September 2002
This paper deals with the microbial production of polyhydroxyalkanoates (PHAs), biodegradable thermoplastics which perform excellently as a material, from inexpensive renewable carbon sources. To date, with the help of genetic engineering techniques, it has become possible to design several types of PHAs with different compositions and to enhance the productivities of PHAs. In addition, molecular breeding of PHA biosynthesis enzymes has been demonstrated to improve polymer production. Mutant PHA synthases generated by an in vitro evolution technique have allowed the enhanced production and quality alteration of PHAs. Furthermore, use of inexpensive renewable carbon sources, such as plant oils, waste materials, and carbon dioxide, would be a key for a reduction in PHA production cost. [Key words: polyhydroxyalkanoates, material property, engineered metabolic pathway, molecular breeding, inexpensive carbon source] Polyhydroxyalkanoates (PHAs) are biological polyesters that are produced by a wide variety of bacteria as an intracellular storage material of carbon and energy. PHAs have recently attracted industrial attention because of their potential use as practical biodegradable and biocompatible thermoplastics. Much research has therefore being focused on the efficient production of PHAs with desirable material properties by wild-type bacteria or recombinants. Hydroxyalkanoate (HA) monomers which form PHA have been broadly divided into two classes, short-chainlength HA (scl-HA) monomers of 3 to 5 carbons, and medium-chain-length HA (mcl-HA) monomers of 6 to 14 carbons. A homopolymer of (R)-3-hydroxybutyrate, P(3HB), is a member of the scl-PHAs and is the most common type of PHA that bacteria accumulate in nature. Although the P(3HB) homopolymer triggered the commercial interest in PHA, naturally occurring P(3HB) does not have sufficient material properties for practical application. Because P(3HB) is a highly crystalline and stiff material, it is brittle and has poor elastic qualities. In contrast, mcl-PHAs are generally regarded as thermoelastomers and rubbers. Recent advances in this area have allowed the production of a new type of copolymer consisting of scl-HA and mcl-HA units using recombinant bacteria. The copolymers are more ductile, easier to mold, and tougher in comparison with the P(3HB) homopolymer. Useful reviews on PHAs have been published by many researchers (l-4). This short review focuses on the recent e-mail: [email protected] phone: +81-(0)45-924-5286
progress in the microbial production of PHAs exhibiting a high performance as plastic materials, and describes their structures and material properties. In addition, molecular breeding of PHA biosynthesis enzymes and use of inexpensive renewable carbon sources for PHA production are discussed with respect to further reduction of the PHA production cost. I.
STRUCTURES AND PROPERTIES OF COMMERCIALLY USEFUL PHAs
P(3HB), containing repeating P(3HB) homopolymer units of (R)-3HB, is the most common biological polyester produced by various bacteria in nature. A gram-negative bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), which is a well-known PHA producer, accumulates P(3HB) up to 80% of the cell dry weight from various carbon sources such as sugars, organic acids, plant oils, and carbon dioxide. P(3HB) isolated from bacteria exhibits 5570% crystallinity, while the P(3HB) molecules in bacteria are amorphous and exist as water insoluble inclusions (2). The weight-average molecular weight (M,) of P(3HB) produced by wild-type bacteria is usually in the range of 1 x 104-3 x lo6 Da with a polydispersity (A&/&Q of around 2.0. The glass-transition temperature (T,) and the melting temperature (r,,,) of the P(3HB) homopolymer are 4°C and 177°C respectively (Table 1). P(3HB) has similar mechanical properties to polypropylene. For example, the tensile strength of P(3HB) (43 MPa) is close to that of polypropylene (38 MPa). However, the extension to break (5%) of P(3HB) is markedly lower than that of polypropylene
fax: +81-(0)45-924-5426 579
580
TSUGE
J. BIOSCI.BIOENG., TABLE 1. Properties of some biosynthesized and chemosynthesized
Polymer P(3HB) Ultra-high-molecular-weight P(3HB) (stretched) P(3HB-co-20 mol% 3HV) P(3HB-co- 16 mol% 4HB) P(3HB-co-10 mol% 3HHx) P(3HB-co-6 mol% 3HA) Polypropylene Low-density polyethylene (LDPE) a Melting temperature. b Glass-transition temperature. c3HA units: 3-hydroxyoctanoate (< 1 mol%).
T,” (“C) 177 185 145 150 127 133 176 130
(cl mol%); 3-hydroxydecanoate
(400%). Therefore, P(3HB) appears as a stiff and more brittle plastic material than the polypropylene. On the other hand, although P(3HB) alone does not have appropriate material properties for practical application, polymer blends of P(3HB) with other biodegradable polymers such as poly(lactide) and poly(s-caprolactone) have improved material properties (5). Ultra-high-molecular-weight P(3HB) Recombinant Escherichia coli harboring R. eutropha PHA biosynthesis genes (phaCAB,) can produce an ultra-high-molecularweight P(3HB) when the culture pH is maintained in the range of 6.0-6.5 (6). This polymer has very high molecular weights (A4,) of 3-20 x 1O6Da. The preparation of P(3HB) stretched films from the ultra-high-molecular-weight P(3HB) has been successful, and it was found that the mechanical properties of the stretched P(3HB) films were markedly improved in comparison with those of the unstretched films
Crystallinity (%) 60 80
(& 4 4 -1 -7 -1 -8 -10 -36
polymers Tensile strength (MPa) 43 400
56 45 34 45 50-70 20-50
(3 mol%); 3-hydroxydodecanoate
20 26 21 17 38 10
50 444 400 680 400 620
(3 mol%); 3-hydroxy-cis-5-dodecanoate
(7, 8). The extension to break and tensile strength of the stretched films increased to 35% and 400 MPa, respectively (Table 1). In addition, when annealing treatment was applied to the stretched films, it was found that the mechanical properties were further improved. Thus, the P(3HB) homopolymer, which was initially a brittle material with poor physical properties, is now a potential candidate for further commercial exploitation with the use of genetic engineering techniques. Copolymers of (R)3HB with HAS As another way to improve the physical properties of P(3HB), the incorporation of different HA units into the P(3HB) sequence to form PHA copolymers is effective. Various bacteria are capable of synthesizing random copolymers of (R)-3HB with other HA units of C3 to C12, depending on both their intrinsic PHA biosynthesis pathways and the carbon sources used. Tables 1 and 2 show the structures of typical PHA
TABLE 2. Microbial synthesis of PHA copolymers containing (R)-3HB as a constituent Bacterial strain
Carbon substrate
Ralstonia eutropha
Propionic acid Pentanoic acid
Aeromonas caviae
Pseudomonas sp. 6 l-3
Random copolymer
(R)-3HB
(R)3HV
(R)3HB
(R)-3HHx
(R)3HB
(R)3HA
Plant oil Fatty acids
Sugar
(n=3-.9)
Ralstonia eutropha Alcaligenes la&s Comamonas acidvorans
Extension to break (%) 5 35
4-Hydroxybutyric acid y-Butyrolactone 1,4-Butanediol 1,6-Hexanediol (R)3HB
4HB
’
VOL.
MICROBIAL PRODUCTION OF PHA
94,2002
copolymers containing the (R)-3HB unit as a constituent, together with their producers. A copolymer of (R)-3HB and (R)-3-hydroxyvalerate [(R)3HV], P(3HB-co-3HV), has been investigated most extensively among the PHA copolymers and applied to commercial products (2). By introducing 20mol% (R)3HV units, the melting temperature and glass-transition temperature of the P(3HB-co-3HV) copolymer decrease to 145°C and -l”C, respectively. In contrast, its extension to break increases to 50%, indicating that the P(3HB-co-3HV) copolymer is more flexible than the P(3HB) homopolymer. Also, the incorporation of 4-hydroxybutyrate (4HB) units into the P(3HB) sequence is more effective in improving the material properties of PHA (2). In the case of (R)-3HB-based copolymers containing (R)-3-hydroxyhexanoate [(R)-3HHx] or (R)3HA (C8-C12), a small amount of the second monomer unit is sufficient to increase their flexibility. It is of interest to note that the P(3HB-co-6 mol% 3HA) copolymer shows similar mechanical properties to low-density polyethylene (LDPE) (9). Thus, this P(3HB-co3H.A) copolymer is expected to have various commercial applications.
Pathway I
Pathway II
Carbonsource &%a@
Acetoacetyl-CoA
Carbon
source
(Fatty acids)
3-Ketoacyl-CoA
(R>3-Hydroxybutyryl-CoA PhaC
1
PHA 4
(R)&Hydroxyacyl-CoA
PhaC
PhaC
t
t 4-Hydmxyacyl-CoA ;* r k,
FabG f’ 3-Ketoacyl-ACP
Other pathway \ t Related carbon sources
PhaG
(R>SHydroxyacyl-ACP
The three best-known Naturally occurring pathways naturally occurring PHA biosynthesis pathways are summarized schematically in Fig. 1. Pathway I, which generates (R)-3HB monomers from acetyl-CoAs, is probably the most common, and has been found in a wide range of bacteria. This pathway has been extensively investigated in Ralstonia eutropha. In R. eutropha, two acetyl-CoA moieties are condensed to yield acetoacetyl-CoA by 3-ketothiolase (PhaA). The product is subsequently reduced to (R)-3HB-CoA by an NADPH-dependent acetoacetyl-CoA reductase (PhaB). Only (R)-isomers are accepted as the substrates of the polymerizing enzyme, PHA synthase (PhaC). Pathways II and III (Fig. l), which generate mainly mcl(R)-3HA monomers from fatty acid P-oxidation intermediates and fatty acid biosynthesis intermediates, respectively, have been found in various fluorescent pseudomonades such as Pseudomonas putida, P oleovorans, and l? aeruginosa. The intermediates in these pathways are effectively converted by some specific enzymes to generate (R)-3HA-CoA monomers for PHA synthase. As shown in Fig. 1, (R)-specitic enoyl-CoA hydratase (PhaJ) and (R)-3-hydroxyacylACP-CoA transferase (PhaG) are capable of supplying (R)3HA-CoA from trans-2-enoyl-CoA and (R)3HA-ACP, respectively. It has been demonstrated that such enzymes are involved in PHA biosynthesis in some bacteria (10, 11). Recently, several other enzymes have been found to possess the ability to supply the monomers. Of significant interest is 3-ketoacyl-ACP reductase (FabG), which is a constituent of the fatty acid biosynthesis pathway. It has been demonstrated that FabG can accept not only acyl-ACP but also acyl-CoA as a substrate and is capable of supplying mcl(R)-3HA-CoA from fatty acid P-oxidation in E. coli (12). To produce PHA copolymers Engineered pathways with desirable monomer compositions, genetic engineering is a very powerful approach. A number of PI-IA synthetic
\
Fatty acid biosynthesis
Enoyl-ACP J
Acyl-ACP t Malonyl-ACP
t Malonyl-CoA t AcetylCoA
II. NATURALLY OCCURRING AND ENGINEERED PHA BIOSYNTHESIS PATHWAYS
581
t
Pathway
Ill
Carbon source (Sugars)
FIG. 1. Metabolic pathways that supply various hydroxyalkanoate (HA) monomers for PHA biosynthesis. PhaA, 3-Ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHA synthase; PhaG, 3-hydroxyacyl-ACP-CoA transferase; PhaJ, @)-specific enoylCoA hydratase; FabG, 3-ketoacyl-ACP reductase.
genes, such as the PHA synthase gene (phaC) and genes encoding monomer supplier (e.g., phaA, phaB, phaG, and pha.j), have been cloned from various PHA-producing bacteria. The products of these genes have inherent substrate specificity. For example, Aeromonas caviae PhaC is active only toward C4, C5 and C6 monomers, whereas Pseudomonas sp. 61-3 PhaC l,, has a broad substrate specificity of C4 to Cl2 monomers. Concerning monomer supply, the R. eutropha PhaAB provide mainly C4 monomers, while PhaJ and PhaG from Pseudomonas strains provide mainly mclmonomers of C6-Cl2 (11, 13). Hence, PHAs with controlled monomer composition can be produced by recombinant bacteria having a combination of the substrate specificities of PHA synthase and monomer-supplying enzyme(s). In addition, the carbon source used also influences the composition. Matsusaki et al. succeeded in the production of a designed copolymer of P(3HB-co-6 mol% 3HA) from glucose, using metabolically engineered Pseudomonas sp. 61-3 (9). The Pseudomonas sp. 61-3 strain (phbC disrupted mutant) produces a P(3HB-co-3HA) random copolymer with a low (R)3HB fraction, which is an elastomer. Hence, to increase the (R)-3HB fraction in the PHA copolymer, the (R)3HB supplying ability of this strain was enhanced by additional expression ofphaAB, genes, resulting in the production of a P(3HB-co-3HA) copolymer with a high (R)-3HB fraction. In contrast, R. eutropha produces a P(3HB) homopolymer from sugar. By functional expression of the phaG gene to supply mcl-(R)-3HA monomers (C8-Cl2) in
582
TSUGE
metabolically engineered R. eutropha (harboring phaCI,,), it has been demonstrated that a new type of P(3HB-co3HA) copolymer can be produced from sugar (14). III. MOLECULAR BREEDING OF PHA BIOSYNTHESIS ENZYMES A new approach in recent PHA rePHA synthase search is the molecular breeding of PHA biosynthesis enzymes by protein engineering techniques. To date, improvements in bacterial PHA production have been achieved through the use of fermentation technology for natural PHAproducing bacteria or the gene dosage effect of PHA biosynthesis genes in recombinant bacteria. Most recently, the enhanced production and quality alteration of PHA have been demonstrated by the application of an in vitro evolution technique to the Aeromonas caviae PHA synthase (PhaC,,) (15). The evolved mutant PhaC,, exhibited higher activity toward the (R)-3HB-CoA monomer than the wildtype enzyme, and consequently led to the enhanced production of the P(3HB-co-3HHx) copolymer from dodecanoate in recombinant E. coli. In addition, an increase in the 3HHx fraction was observed for the evolved mutant relative to the wild-type enzyme. Also, the in vitro evolution technique was applied to the R. eutropha PHA synthase (PhaC,,), resulting in the generation of highly active mutants (16). At present, although there is no available X-ray crystal structure of PHA synthase, the in vitro evolution technique is a very useful approach to achieving enzyme improvement. Monomer-supplying enzymes In contrast to PHA synthase, there are several available X-ray crystal structures of enzymes homologous to monomer suppliers in the PHA biosynthesis pathway. For example, the crystal structure of the Zoogloea ramigera 3-ketothiolase, which exhibits a high similarity to PhaA,, has already been solved (17) and its tertiary structural information is now available through the internet. In our group, we are undertaking the crystal structure analysis of Phal,, (18). As well as PHA synthase, the molecular breeding of monomer-supplying enzymes would be important for the further development of microbial PHA production. Hence, the available tertiary structural information will help us to improve the property of monomer suppliers in terms of substrate specificity and enzyme activity by using site-directed mutagenesis or other techniques. IV.
USE OF INEXPENSIVE CARBON SOURCES FOR PHA PRODUCTION
To date, PHA proConventional PHA production duction by wild-type strains and recombinants is usually performed in two-stage fed-batch cultures, which consist of a cell-growth phase and a PHA-production phase. In the cell-growth phase, nutritionally enriched medium is used to obtain high cell mass during early cultivation. In the sequential PHA-production phase, the cell growth is limited by depletion of some nutrients such as nitrogen, phosphorus, oxygen, or magnesium (4). This depletion acts as a trigger for the metabolic shift to PHA biosynthesis. Sugars such as glucose and sucrose are the most common
J. BIOSCI. BIOENG.,
main carbon sources for PHA production because they can be obtained at a relatively low price. The highest P(3HB) productivity from glucose was obtained by a recombinant E. coli harboring Alcaligenes latus PHA biosynthesis genes @haCAB,,) (19). In this culture, P(3HB) productivity of 4.63 gl2.h was obtained, and the P(3HB) concentration and P(3HB) content in the dry cells reached 142 g/Z, and 73%, respectively, in 30 h. For the production of P(3HB-co-3HV) copolymer, propionate or pentanoate is usually added to the medium as a co-substrate for providing the precursors of (R)-3HV units, The highest copolymer productivity of 2.88 gl1.h was also obtained using the recombinant E. coli harboring A. latus PHA biosynthesis genes (20). Use of more inexpensive carbon sources Until recently, the P(3HB-co-3HV) copolymer had been produced from glucose and propionate on a semi-commercial scale first by Zeneca BioProducts (Billingham, UK), and later by Monsanto (St. Louis, MO, USA). Although the cost of polyolelins is less than US $1 kg-‘, the price of PHA is much higher than that of synthetic plastic. The price of PHA will be further reduced by the use of more inexpensive carbon sources. Lee et al. reported that P(3HB) and mcl-PHA can be produced at a cost of approximately US $2 kg-’ by an effective production strategy to attain a high PHA productivity and PHA concentration using an inexpensive carbon source (21). Since PHA production from pure glucose or sucrose has already been optimized, the development of fermentation technology to use cheaper carbon sources would be a key factor in further reducing the PHA production cost. Plant oils or their derived Plant oils or fatty acids fatty acids are good carbon sources for PHA production because they are inexpensive renewable carbon sources. In addition, the theoretical yield coefficient of PHA fi-om fatty acid (e.g., 0.65-0.98 kg/kg from butyric acid) is considerably higher than that from glucose (0.32-0.48 kg/kg) (22). However, bacterial fermentation using plant oils or fatty acids still has several problems. One major problem is the relatively low growth rate of available PHA-producing bacteria. Even if a bacterium, the growth rate of which on plant oils or fatty acids is rather high, is employed for PHA production, the PHA content in the dry cells is relatively low. Indeed, a large-scale production of P(3HB-co-3HHx) from lauric acid was carried out using Aeromonas hydrophila with a final PHA content of 50 wt% (23). More research on the breeding of superior plant oil-utilizing strains or on the development of fermentation technology for using plant oils is needed to overcome these problems. Agricultural or food industrial wastes Agricultural or food industrial waste materials can be used as inexpensive carbon and nitrogen sources for PHA-producing bacteria. Xylose is the second most abundant sugar in such waste materials, but efficient PHA production from xylose is difficult due to its low assimilation rate by PHA-producing bacteria. Lactic acid and acetic acid, obtained from xylose by an anaerobic fermentation of lactic acid bacteria, are feasible carbon sources for PHA production, because most PHA-producing bacteria can assimilate them at a high rate. Indeed, high P(3HB) productivities of 1.1 g/Z. h and 1.3 gll. h were obtained using lactic acid and a lactic acid/acetic acid mixture, respectively, as carbon sources for R. eutropha
VOL.94,2002
MICROBIAL
(24, 25). Also, other organic acids generated by anaerobic fermentation can be used as carbon sources for PHA production (26). Carbon dioxide Carbon dioxide in the atmosphere is the ultimate feedstock for PHA production. Cyanobacteria and some photosynthetic bacteria can immediately assimilate carbon dioxide from the atmosphere using light energy. Some wild-type cyanobacteria are capable of accumulating small amounts of P(3HB) (approximately 6 wt%) in the cells from carbon dioxide. Miyake et al. found that the thermophilic cyanobacterium Synechococcus sp. MA19 is capable of accumulating up to 27 wt% of PHA in cells from carbon dioxide (27). Further investigation demonstrated that strain MA19 can accumulate 55 wt% of P(3HB) in the cells under optimized culture conditions (28). In the near future, cyanobacteria will be used as good producers of PHA. In contrast, R. eutropha can assimilate carbon dioxide and produce P(3HB) in the absence of light energy, but with oxidization of hydrogen (29-32). To date, hydrogen has attracted industrial attention as a clean energy source and an alternative to petroleum. If a process for providing a large amount of hydrogen at a low price is developed, PHA production from carbon dioxide by R. eutropha would be improved, because the P(3HB) productivity of R. eutropha (1.55 gl1.h) is considerably higher than those of other bacteria such as cyanobacteria and photosynthetic bacteria (32). CONCLUSION PHAs have been considered to be good candidates for biodegradable thermoplastics, but it is essential to improve their material properties and to reduce the production cost if they are to be used as bulk products. With the help of genetic and protein engineering, it has been possible to design several types of PHAs and to enhance the polymer productivity. For example, recombinant E. coli is capable of producing ultra-high-molecular-weight P(3HB), which has superior properties after stretching of the material. Metabolically engineered Pseudomonas sp. 61-3 produces P(3HBco-3HA) copolymer, the material properties of which are similar to those of low-density polyethylene. In addition, application of the in vitro evolution technique to PHA synthase led to the generation of a designed strain exhibiting the enhanced polymer production. On the other hand, for PHA production, use of inexpensive renewable carbon sources such as plant oils, waste materials, and carbon dioxide is essential to reduce the production cost further. In the near future, all of these efforts will overcome the current problems concerning the production and application of PHAs. ACKOWLEDGMENT I would like to thank Prof. Y. Doi (Tokyo Institute of Technology, Yokohama, and RlKEN Institute, Saitama) for many fruitful discussions and for help with the preparation of this manuscript.
REFERENCES 1. Sudesh, K., Abe, H., and Doi, Y.: Synthesis,
structure and
properties 2. 3.
4.
5.
PRODUCTION
of polyhydroxyalkanoates:
OF PHA
biological
583
polyesters.
Proe. Polvm. Sci.. 25. 1503-1555 120001. Do[ Y.: ‘Microbial iolymers. V&l Piblishers, New York (1990). Doi, Y. and Steinbiichel, A. (ea.): Polyester I, biological systems and biotechnological production. Wiley-VCH, Weinheim (2002). Madison, L. L. and Huisman, G. W.: Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev., 63,21-53 (1999). Urakami, T.: Biogreen. Plastics, 50, 107-111 (1999). (in
Japanese) 6. Kusaka, S., Abe, H., Lee, S. Y., and Doi, Y.: Molecular mass of poly[(R)-3-hydroxybutyric acid] produced in a recombinant Escherichia cob. Appl. Microbial. Biotechnol.,
47, 140-143 (1997). S., Iwata, T., and Doi, Y.: Properties and biodegradability of ultra-high-molecular-weight poly[(R)-hydroxybutyrate] produced by a recombinant Escherichia coli. Int. J. Biol. Macromol., 25, 87-94 (1999).
7. Kusaka,
8. Kusaka, S., Iwata, T., and Doi, Y.: Microbial synthesis and properties of ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate]. J. Macromol. Sci. Pure Appl. Chem. A, 35, 319335 (1998). 9. Matsusaki, H., Abe, H., and Doi, Y.: Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant strains of Pseudomonas sp. 6 l-3. Biomacromolecules, 1, 17-22 (2000). 10. Fukui, T., Shiomi, N., and Doi, Y.: Expression and characterization of (R)-specific enoyl coenzyme A hydra&e involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae. J. Bacterial., 180, 667473 (1998). 11. Rehm, B. H., Kruger, N., and Steinbiichel, A.: A new metabolic link between fatty acid de nova synthesis and polyhydroxyalkanoic acid synthesis. The phaG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrier protein-coenzyme A transferase. J. Biol. Chem., 273, 24044-2405 1 (1998). 12. Taguchi, K., Aoyagi, Y., Matsusaki, H., Fukui, T., and Doi, Y.: Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HBlOl strain. FEMS Microbial. Lett., 176,
183-190 (1999). 13. Tsuge, T., Fukui, T., Matsusaki, H., Taguchi, S., Kobayashi, G., Ishizaki, A., and Doi, Y.: Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas ueruginosa and their use for polyhydroxyalkanoate
synthesis. FEMS Microbial. Lett., 184, 193-198 (2000). 14. Matsumoto, K., Nakae, S., Taguchi, K., Matsusaki, H., Seki, M., and Doi, Y.: Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) copolymer from sugars by recombinant Rulstoniu eutrophu harboring the phuCI,, and the phuG, genes of Pseudomonas sp. 61-3. Biomacromolecules, 2, 934-939 (2001). 15. Kichise, T., Taguchi, S., and Doi, Y.: Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromenus cuviae PHA synthase. Appl. Environ. Microbial., 68, 2411-2419 (2002). 16. Taguchi, S., Nakamura, H., Hiraishi, T., Yamato, I., and Doi, Y.: In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis. J. Biochem. (Tokyo), 131,801-806 (2002). 17. Modis, Y. and Wierenga, R. K.: Crystallographic analysis of the reaction pathway of Zoogloeu rumigera biosynthetic thiolase. J. Mol. Biol., 297, 1171-l 182 (2000). 18. Hisano, T., Fukui, T., Iwata, T., and Doi, Y.: Crystallization and preliminary X-ray analysis of (R)-specific enoyl-CoA hydratase from Aeromonus cuviae involved in polyhydroxy-
584
J. BIOSCI.BIOENG.,
TSUGE
alkanoate biosynthesis. Acta Crystallogr. D. Biol. Crystallogr., 57, 145-147 (2001). 19. Cboi, J. I., Lee, S. Y., and Han, K.: Cloning of the AZcaZigenes labs polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of poly(3-hydroxybutyrate) in Escherichia coli. Appl. Environ. Microbial., 64, 48974903 (1998). 20. Choi, J. I. and Lee, S. Y.: High-level production of poly(3hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli. Avvl. Environ. Microbial..
27.
28.
1.
65,4363-4368 (1999). 21. Lee, S.Y., Choi, J., and Wong, H.H.: Recent advances in polyhydroxyalkanoate production by bacterial fermentation. Int. J. Biol. Macromol., 25,31-36 (1999). 22. Yamane, T.: Cultivation engineering of microbial bioplastics production. FEMS Microbial. Rev., 103,257-264 (1992). 23. Chen, G. Q., Zhang, G., Park, S. J., and Lee, S. Y.: Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Appl. Microbial. Biotechnol., 57, 50-55 (2001). 24. Tsuge, T., Tanaka, K., and Ishizaki, A.: Development of a novel method for feeding a mixture of L-lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly-D-3hydroxybutyrate production. J. Biosci. Bioeng., 91, 545-550 (2001). 25. Tsuge, T,, Tanaka, K., Shimoda, M., and Ishizaki, A.: Optimization of L-lactic acid feeding for production of poly-D-3hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture. J. Biosci. Bioeng., 88,404-409 (1999). 26. Kohayashi, G., Tanaka, K., Itoh, I-I., Tsuge, T., Sonomoto,
29.
30.
3 1.
32.
K., and Ishizaki, A.: Fermentative production of P(3HBco3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method. Biotechnol. Lett., 22, 1067-1069 (2000). Miyake, M., Erata, M., and Asada, Y.: A thermophilic cyanobacterium, Synechococcus sp. MA19, capable of accumulating poly+hydroxybutyrate. J. Ferment. Bioeng., 82, 512-514 (1996). Nishioka, M., Nakai, K., Miyake, M., Asada, Y., and Taya, M.: Production of the poly-j3-hydroxyalkanoate by thermophilic cyanobacterium, S’echococcus sp. MA19, under phosphate-limitted condition. Biotechnol. Lett., 23, 1095-1099 (2001). Ishizaki, A., Tanaka, K., and Taga, N.: Microbial production of poly-D-3-hydroxybutyrate from CO,. Appl. Microbial. Biotechnol., 57,6-12 (2001). Ishizaki, A. and Tanaka, K.: Production of poly+hydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC176977 J. Ferment. Bioeng., 71,254-257 (1991). Sugimoto, T., Tsuge, T., Tanaka, K., and Ishizaki, A.: Control of acetic acid concentration by pH-stat continuous substrate feeding in heterotrophic culture phase of two-stage cultivation of Alcaligenes eutrophus for production of P(3HB) from CO,, H,, and 0, under non-explosive conditions. Biotechnol. Bioeng., 62,625-631 (1999). Tanaka, K., Ishizaki, A., Kanamaru, T., and Kawano, T.: Production of poly(D-3-hydroxybutyrate) from CO,, H,, and 0, by high cell density autotrophic cultivation of Alcaligenes eutrophus. Biotechnol. Bioeng., 45,268-275 (1995).
REVIEW Metabolic Improvements and Use of Inexpensive Carbon Sources in Microbial Production of Polyhydroxyalkanoates TAKEHARU
TSUGE’
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan’ Received 23 July 2002IAccepted
4 September 2002
This paper deals with the microbial production of polyhydroxyalkanoates (PHAs), biodegradable thermoplastics which perform excellently as a material, from inexpensive renewable carbon sources. To date, with the help of genetic engineering techniques, it has become possible to design several types of PHAs with different compositions and to enhance the productivities of PHAs. In addition, molecular breeding of PHA biosynthesis enzymes has been demonstrated to improve polymer production. Mutant PHA synthases generated by an in vitro evolution technique have allowed the enhanced production and quality alteration of PHAs. Furthermore, use of inexpensive renewable carbon sources, such as plant oils, waste materials, and carbon dioxide, would be a key for a reduction in PHA production cost. [Key words: polyhydroxyalkanoates, material property, engineered metabolic pathway, molecular breeding, inexpensive carbon source] Polyhydroxyalkanoates (PHAs) are biological polyesters that are produced by a wide variety of bacteria as an intracellular storage material of carbon and energy. PHAs have recently attracted industrial attention because of their potential use as practical biodegradable and biocompatible thermoplastics. Much research has therefore being focused on the efficient production of PHAs with desirable material properties by wild-type bacteria or recombinants. Hydroxyalkanoate (HA) monomers which form PHA have been broadly divided into two classes, short-chainlength HA (scl-HA) monomers of 3 to 5 carbons, and medium-chain-length HA (mcl-HA) monomers of 6 to 14 carbons. A homopolymer of (R)-3-hydroxybutyrate, P(3HB), is a member of the scl-PHAs and is the most common type of PHA that bacteria accumulate in nature. Although the P(3HB) homopolymer triggered the commercial interest in PHA, naturally occurring P(3HB) does not have sufficient material properties for practical application. Because P(3HB) is a highly crystalline and stiff material, it is brittle and has poor elastic qualities. In contrast, mcl-PHAs are generally regarded as thermoelastomers and rubbers. Recent advances in this area have allowed the production of a new type of copolymer consisting of scl-HA and mcl-HA units using recombinant bacteria. The copolymers are more ductile, easier to mold, and tougher in comparison with the P(3HB) homopolymer. Useful reviews on PHAs have been published by many researchers (l-4). This short review focuses on the recent e-mail: [email protected] phone: +81-(0)45-924-5286
progress in the microbial production of PHAs exhibiting a high performance as plastic materials, and describes their structures and material properties. In addition, molecular breeding of PHA biosynthesis enzymes and use of inexpensive renewable carbon sources for PHA production are discussed with respect to further reduction of the PHA production cost. I.
STRUCTURES AND PROPERTIES OF COMMERCIALLY USEFUL PHAs
P(3HB), containing repeating P(3HB) homopolymer units of (R)-3HB, is the most common biological polyester produced by various bacteria in nature. A gram-negative bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), which is a well-known PHA producer, accumulates P(3HB) up to 80% of the cell dry weight from various carbon sources such as sugars, organic acids, plant oils, and carbon dioxide. P(3HB) isolated from bacteria exhibits 5570% crystallinity, while the P(3HB) molecules in bacteria are amorphous and exist as water insoluble inclusions (2). The weight-average molecular weight (M,) of P(3HB) produced by wild-type bacteria is usually in the range of 1 x 104-3 x lo6 Da with a polydispersity (A&/&Q of around 2.0. The glass-transition temperature (T,) and the melting temperature (r,,,) of the P(3HB) homopolymer are 4°C and 177°C respectively (Table 1). P(3HB) has similar mechanical properties to polypropylene. For example, the tensile strength of P(3HB) (43 MPa) is close to that of polypropylene (38 MPa). However, the extension to break (5%) of P(3HB) is markedly lower than that of polypropylene
fax: +81-(0)45-924-5426 579
580
TSUGE
J. BIOSCI.BIOENG., TABLE 1. Properties of some biosynthesized and chemosynthesized
Polymer P(3HB) Ultra-high-molecular-weight P(3HB) (stretched) P(3HB-co-20 mol% 3HV) P(3HB-co- 16 mol% 4HB) P(3HB-co-10 mol% 3HHx) P(3HB-co-6 mol% 3HA) Polypropylene Low-density polyethylene (LDPE) a Melting temperature. b Glass-transition temperature. c3HA units: 3-hydroxyoctanoate (< 1 mol%).
T,” (“C) 177 185 145 150 127 133 176 130
(cl mol%); 3-hydroxydecanoate
(400%). Therefore, P(3HB) appears as a stiff and more brittle plastic material than the polypropylene. On the other hand, although P(3HB) alone does not have appropriate material properties for practical application, polymer blends of P(3HB) with other biodegradable polymers such as poly(lactide) and poly(s-caprolactone) have improved material properties (5). Ultra-high-molecular-weight P(3HB) Recombinant Escherichia coli harboring R. eutropha PHA biosynthesis genes (phaCAB,) can produce an ultra-high-molecularweight P(3HB) when the culture pH is maintained in the range of 6.0-6.5 (6). This polymer has very high molecular weights (A4,) of 3-20 x 1O6Da. The preparation of P(3HB) stretched films from the ultra-high-molecular-weight P(3HB) has been successful, and it was found that the mechanical properties of the stretched P(3HB) films were markedly improved in comparison with those of the unstretched films
Crystallinity (%) 60 80
(& 4 4 -1 -7 -1 -8 -10 -36
polymers Tensile strength (MPa) 43 400
56 45 34 45 50-70 20-50
(3 mol%); 3-hydroxydodecanoate
20 26 21 17 38 10
50 444 400 680 400 620
(3 mol%); 3-hydroxy-cis-5-dodecanoate
(7, 8). The extension to break and tensile strength of the stretched films increased to 35% and 400 MPa, respectively (Table 1). In addition, when annealing treatment was applied to the stretched films, it was found that the mechanical properties were further improved. Thus, the P(3HB) homopolymer, which was initially a brittle material with poor physical properties, is now a potential candidate for further commercial exploitation with the use of genetic engineering techniques. Copolymers of (R)3HB with HAS As another way to improve the physical properties of P(3HB), the incorporation of different HA units into the P(3HB) sequence to form PHA copolymers is effective. Various bacteria are capable of synthesizing random copolymers of (R)-3HB with other HA units of C3 to C12, depending on both their intrinsic PHA biosynthesis pathways and the carbon sources used. Tables 1 and 2 show the structures of typical PHA
TABLE 2. Microbial synthesis of PHA copolymers containing (R)-3HB as a constituent Bacterial strain
Carbon substrate
Ralstonia eutropha
Propionic acid Pentanoic acid
Aeromonas caviae
Pseudomonas sp. 6 l-3
Random copolymer
(R)-3HB
(R)3HV
(R)3HB
(R)-3HHx
(R)3HB
(R)3HA
Plant oil Fatty acids
Sugar
(n=3-.9)
Ralstonia eutropha Alcaligenes la&s Comamonas acidvorans
Extension to break (%) 5 35
4-Hydroxybutyric acid y-Butyrolactone 1,4-Butanediol 1,6-Hexanediol (R)3HB
4HB
’
VOL.
MICROBIAL PRODUCTION OF PHA
94,2002
copolymers containing the (R)-3HB unit as a constituent, together with their producers. A copolymer of (R)-3HB and (R)-3-hydroxyvalerate [(R)3HV], P(3HB-co-3HV), has been investigated most extensively among the PHA copolymers and applied to commercial products (2). By introducing 20mol% (R)3HV units, the melting temperature and glass-transition temperature of the P(3HB-co-3HV) copolymer decrease to 145°C and -l”C, respectively. In contrast, its extension to break increases to 50%, indicating that the P(3HB-co-3HV) copolymer is more flexible than the P(3HB) homopolymer. Also, the incorporation of 4-hydroxybutyrate (4HB) units into the P(3HB) sequence is more effective in improving the material properties of PHA (2). In the case of (R)-3HB-based copolymers containing (R)-3-hydroxyhexanoate [(R)-3HHx] or (R)3HA (C8-C12), a small amount of the second monomer unit is sufficient to increase their flexibility. It is of interest to note that the P(3HB-co-6 mol% 3HA) copolymer shows similar mechanical properties to low-density polyethylene (LDPE) (9). Thus, this P(3HB-co3H.A) copolymer is expected to have various commercial applications.
Pathway I
Pathway II
Carbonsource &%a@
Acetoacetyl-CoA
Carbon
source
(Fatty acids)
3-Ketoacyl-CoA
(R>3-Hydroxybutyryl-CoA PhaC
1
PHA 4
(R)&Hydroxyacyl-CoA
PhaC
PhaC
t
t 4-Hydmxyacyl-CoA ;* r k,
FabG f’ 3-Ketoacyl-ACP
Other pathway \ t Related carbon sources
PhaG
(R>SHydroxyacyl-ACP
The three best-known Naturally occurring pathways naturally occurring PHA biosynthesis pathways are summarized schematically in Fig. 1. Pathway I, which generates (R)-3HB monomers from acetyl-CoAs, is probably the most common, and has been found in a wide range of bacteria. This pathway has been extensively investigated in Ralstonia eutropha. In R. eutropha, two acetyl-CoA moieties are condensed to yield acetoacetyl-CoA by 3-ketothiolase (PhaA). The product is subsequently reduced to (R)-3HB-CoA by an NADPH-dependent acetoacetyl-CoA reductase (PhaB). Only (R)-isomers are accepted as the substrates of the polymerizing enzyme, PHA synthase (PhaC). Pathways II and III (Fig. l), which generate mainly mcl(R)-3HA monomers from fatty acid P-oxidation intermediates and fatty acid biosynthesis intermediates, respectively, have been found in various fluorescent pseudomonades such as Pseudomonas putida, P oleovorans, and l? aeruginosa. The intermediates in these pathways are effectively converted by some specific enzymes to generate (R)-3HA-CoA monomers for PHA synthase. As shown in Fig. 1, (R)-specitic enoyl-CoA hydratase (PhaJ) and (R)-3-hydroxyacylACP-CoA transferase (PhaG) are capable of supplying (R)3HA-CoA from trans-2-enoyl-CoA and (R)3HA-ACP, respectively. It has been demonstrated that such enzymes are involved in PHA biosynthesis in some bacteria (10, 11). Recently, several other enzymes have been found to possess the ability to supply the monomers. Of significant interest is 3-ketoacyl-ACP reductase (FabG), which is a constituent of the fatty acid biosynthesis pathway. It has been demonstrated that FabG can accept not only acyl-ACP but also acyl-CoA as a substrate and is capable of supplying mcl(R)-3HA-CoA from fatty acid P-oxidation in E. coli (12). To produce PHA copolymers Engineered pathways with desirable monomer compositions, genetic engineering is a very powerful approach. A number of PI-IA synthetic
\
Fatty acid biosynthesis
Enoyl-ACP J
Acyl-ACP t Malonyl-ACP
t Malonyl-CoA t AcetylCoA
II. NATURALLY OCCURRING AND ENGINEERED PHA BIOSYNTHESIS PATHWAYS
581
t
Pathway
Ill
Carbon source (Sugars)
FIG. 1. Metabolic pathways that supply various hydroxyalkanoate (HA) monomers for PHA biosynthesis. PhaA, 3-Ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHA synthase; PhaG, 3-hydroxyacyl-ACP-CoA transferase; PhaJ, @)-specific enoylCoA hydratase; FabG, 3-ketoacyl-ACP reductase.
genes, such as the PHA synthase gene (phaC) and genes encoding monomer supplier (e.g., phaA, phaB, phaG, and pha.j), have been cloned from various PHA-producing bacteria. The products of these genes have inherent substrate specificity. For example, Aeromonas caviae PhaC is active only toward C4, C5 and C6 monomers, whereas Pseudomonas sp. 61-3 PhaC l,, has a broad substrate specificity of C4 to Cl2 monomers. Concerning monomer supply, the R. eutropha PhaAB provide mainly C4 monomers, while PhaJ and PhaG from Pseudomonas strains provide mainly mclmonomers of C6-Cl2 (11, 13). Hence, PHAs with controlled monomer composition can be produced by recombinant bacteria having a combination of the substrate specificities of PHA synthase and monomer-supplying enzyme(s). In addition, the carbon source used also influences the composition. Matsusaki et al. succeeded in the production of a designed copolymer of P(3HB-co-6 mol% 3HA) from glucose, using metabolically engineered Pseudomonas sp. 61-3 (9). The Pseudomonas sp. 61-3 strain (phbC disrupted mutant) produces a P(3HB-co-3HA) random copolymer with a low (R)3HB fraction, which is an elastomer. Hence, to increase the (R)-3HB fraction in the PHA copolymer, the (R)3HB supplying ability of this strain was enhanced by additional expression ofphaAB, genes, resulting in the production of a P(3HB-co-3HA) copolymer with a high (R)-3HB fraction. In contrast, R. eutropha produces a P(3HB) homopolymer from sugar. By functional expression of the phaG gene to supply mcl-(R)-3HA monomers (C8-Cl2) in
582
TSUGE
metabolically engineered R. eutropha (harboring phaCI,,), it has been demonstrated that a new type of P(3HB-co3HA) copolymer can be produced from sugar (14). III. MOLECULAR BREEDING OF PHA BIOSYNTHESIS ENZYMES A new approach in recent PHA rePHA synthase search is the molecular breeding of PHA biosynthesis enzymes by protein engineering techniques. To date, improvements in bacterial PHA production have been achieved through the use of fermentation technology for natural PHAproducing bacteria or the gene dosage effect of PHA biosynthesis genes in recombinant bacteria. Most recently, the enhanced production and quality alteration of PHA have been demonstrated by the application of an in vitro evolution technique to the Aeromonas caviae PHA synthase (PhaC,,) (15). The evolved mutant PhaC,, exhibited higher activity toward the (R)-3HB-CoA monomer than the wildtype enzyme, and consequently led to the enhanced production of the P(3HB-co-3HHx) copolymer from dodecanoate in recombinant E. coli. In addition, an increase in the 3HHx fraction was observed for the evolved mutant relative to the wild-type enzyme. Also, the in vitro evolution technique was applied to the R. eutropha PHA synthase (PhaC,,), resulting in the generation of highly active mutants (16). At present, although there is no available X-ray crystal structure of PHA synthase, the in vitro evolution technique is a very useful approach to achieving enzyme improvement. Monomer-supplying enzymes In contrast to PHA synthase, there are several available X-ray crystal structures of enzymes homologous to monomer suppliers in the PHA biosynthesis pathway. For example, the crystal structure of the Zoogloea ramigera 3-ketothiolase, which exhibits a high similarity to PhaA,, has already been solved (17) and its tertiary structural information is now available through the internet. In our group, we are undertaking the crystal structure analysis of Phal,, (18). As well as PHA synthase, the molecular breeding of monomer-supplying enzymes would be important for the further development of microbial PHA production. Hence, the available tertiary structural information will help us to improve the property of monomer suppliers in terms of substrate specificity and enzyme activity by using site-directed mutagenesis or other techniques. IV.
USE OF INEXPENSIVE CARBON SOURCES FOR PHA PRODUCTION
To date, PHA proConventional PHA production duction by wild-type strains and recombinants is usually performed in two-stage fed-batch cultures, which consist of a cell-growth phase and a PHA-production phase. In the cell-growth phase, nutritionally enriched medium is used to obtain high cell mass during early cultivation. In the sequential PHA-production phase, the cell growth is limited by depletion of some nutrients such as nitrogen, phosphorus, oxygen, or magnesium (4). This depletion acts as a trigger for the metabolic shift to PHA biosynthesis. Sugars such as glucose and sucrose are the most common
J. BIOSCI. BIOENG.,
main carbon sources for PHA production because they can be obtained at a relatively low price. The highest P(3HB) productivity from glucose was obtained by a recombinant E. coli harboring Alcaligenes latus PHA biosynthesis genes @haCAB,,) (19). In this culture, P(3HB) productivity of 4.63 gl2.h was obtained, and the P(3HB) concentration and P(3HB) content in the dry cells reached 142 g/Z, and 73%, respectively, in 30 h. For the production of P(3HB-co-3HV) copolymer, propionate or pentanoate is usually added to the medium as a co-substrate for providing the precursors of (R)-3HV units, The highest copolymer productivity of 2.88 gl1.h was also obtained using the recombinant E. coli harboring A. latus PHA biosynthesis genes (20). Use of more inexpensive carbon sources Until recently, the P(3HB-co-3HV) copolymer had been produced from glucose and propionate on a semi-commercial scale first by Zeneca BioProducts (Billingham, UK), and later by Monsanto (St. Louis, MO, USA). Although the cost of polyolelins is less than US $1 kg-‘, the price of PHA is much higher than that of synthetic plastic. The price of PHA will be further reduced by the use of more inexpensive carbon sources. Lee et al. reported that P(3HB) and mcl-PHA can be produced at a cost of approximately US $2 kg-’ by an effective production strategy to attain a high PHA productivity and PHA concentration using an inexpensive carbon source (21). Since PHA production from pure glucose or sucrose has already been optimized, the development of fermentation technology to use cheaper carbon sources would be a key factor in further reducing the PHA production cost. Plant oils or their derived Plant oils or fatty acids fatty acids are good carbon sources for PHA production because they are inexpensive renewable carbon sources. In addition, the theoretical yield coefficient of PHA fi-om fatty acid (e.g., 0.65-0.98 kg/kg from butyric acid) is considerably higher than that from glucose (0.32-0.48 kg/kg) (22). However, bacterial fermentation using plant oils or fatty acids still has several problems. One major problem is the relatively low growth rate of available PHA-producing bacteria. Even if a bacterium, the growth rate of which on plant oils or fatty acids is rather high, is employed for PHA production, the PHA content in the dry cells is relatively low. Indeed, a large-scale production of P(3HB-co-3HHx) from lauric acid was carried out using Aeromonas hydrophila with a final PHA content of 50 wt% (23). More research on the breeding of superior plant oil-utilizing strains or on the development of fermentation technology for using plant oils is needed to overcome these problems. Agricultural or food industrial wastes Agricultural or food industrial waste materials can be used as inexpensive carbon and nitrogen sources for PHA-producing bacteria. Xylose is the second most abundant sugar in such waste materials, but efficient PHA production from xylose is difficult due to its low assimilation rate by PHA-producing bacteria. Lactic acid and acetic acid, obtained from xylose by an anaerobic fermentation of lactic acid bacteria, are feasible carbon sources for PHA production, because most PHA-producing bacteria can assimilate them at a high rate. Indeed, high P(3HB) productivities of 1.1 g/Z. h and 1.3 gll. h were obtained using lactic acid and a lactic acid/acetic acid mixture, respectively, as carbon sources for R. eutropha
VOL.94,2002
MICROBIAL
(24, 25). Also, other organic acids generated by anaerobic fermentation can be used as carbon sources for PHA production (26). Carbon dioxide Carbon dioxide in the atmosphere is the ultimate feedstock for PHA production. Cyanobacteria and some photosynthetic bacteria can immediately assimilate carbon dioxide from the atmosphere using light energy. Some wild-type cyanobacteria are capable of accumulating small amounts of P(3HB) (approximately 6 wt%) in the cells from carbon dioxide. Miyake et al. found that the thermophilic cyanobacterium Synechococcus sp. MA19 is capable of accumulating up to 27 wt% of PHA in cells from carbon dioxide (27). Further investigation demonstrated that strain MA19 can accumulate 55 wt% of P(3HB) in the cells under optimized culture conditions (28). In the near future, cyanobacteria will be used as good producers of PHA. In contrast, R. eutropha can assimilate carbon dioxide and produce P(3HB) in the absence of light energy, but with oxidization of hydrogen (29-32). To date, hydrogen has attracted industrial attention as a clean energy source and an alternative to petroleum. If a process for providing a large amount of hydrogen at a low price is developed, PHA production from carbon dioxide by R. eutropha would be improved, because the P(3HB) productivity of R. eutropha (1.55 gl1.h) is considerably higher than those of other bacteria such as cyanobacteria and photosynthetic bacteria (32). CONCLUSION PHAs have been considered to be good candidates for biodegradable thermoplastics, but it is essential to improve their material properties and to reduce the production cost if they are to be used as bulk products. With the help of genetic and protein engineering, it has been possible to design several types of PHAs and to enhance the polymer productivity. For example, recombinant E. coli is capable of producing ultra-high-molecular-weight P(3HB), which has superior properties after stretching of the material. Metabolically engineered Pseudomonas sp. 61-3 produces P(3HBco-3HA) copolymer, the material properties of which are similar to those of low-density polyethylene. In addition, application of the in vitro evolution technique to PHA synthase led to the generation of a designed strain exhibiting the enhanced polymer production. On the other hand, for PHA production, use of inexpensive renewable carbon sources such as plant oils, waste materials, and carbon dioxide is essential to reduce the production cost further. In the near future, all of these efforts will overcome the current problems concerning the production and application of PHAs. ACKOWLEDGMENT I would like to thank Prof. Y. Doi (Tokyo Institute of Technology, Yokohama, and RlKEN Institute, Saitama) for many fruitful discussions and for help with the preparation of this manuscript.
REFERENCES 1. Sudesh, K., Abe, H., and Doi, Y.: Synthesis,
structure and
properties 2. 3.
4.
5.
PRODUCTION
of polyhydroxyalkanoates:
OF PHA
biological
583
polyesters.
Proe. Polvm. Sci.. 25. 1503-1555 120001. Do[ Y.: ‘Microbial iolymers. V&l Piblishers, New York (1990). Doi, Y. and Steinbiichel, A. (ea.): Polyester I, biological systems and biotechnological production. Wiley-VCH, Weinheim (2002). Madison, L. L. and Huisman, G. W.: Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev., 63,21-53 (1999). Urakami, T.: Biogreen. Plastics, 50, 107-111 (1999). (in
Japanese) 6. Kusaka, S., Abe, H., Lee, S. Y., and Doi, Y.: Molecular mass of poly[(R)-3-hydroxybutyric acid] produced in a recombinant Escherichia cob. Appl. Microbial. Biotechnol.,
47, 140-143 (1997). S., Iwata, T., and Doi, Y.: Properties and biodegradability of ultra-high-molecular-weight poly[(R)-hydroxybutyrate] produced by a recombinant Escherichia coli. Int. J. Biol. Macromol., 25, 87-94 (1999).
7. Kusaka,
8. Kusaka, S., Iwata, T., and Doi, Y.: Microbial synthesis and properties of ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate]. J. Macromol. Sci. Pure Appl. Chem. A, 35, 319335 (1998). 9. Matsusaki, H., Abe, H., and Doi, Y.: Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant strains of Pseudomonas sp. 6 l-3. Biomacromolecules, 1, 17-22 (2000). 10. Fukui, T., Shiomi, N., and Doi, Y.: Expression and characterization of (R)-specific enoyl coenzyme A hydra&e involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae. J. Bacterial., 180, 667473 (1998). 11. Rehm, B. H., Kruger, N., and Steinbiichel, A.: A new metabolic link between fatty acid de nova synthesis and polyhydroxyalkanoic acid synthesis. The phaG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrier protein-coenzyme A transferase. J. Biol. Chem., 273, 24044-2405 1 (1998). 12. Taguchi, K., Aoyagi, Y., Matsusaki, H., Fukui, T., and Doi, Y.: Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HBlOl strain. FEMS Microbial. Lett., 176,
183-190 (1999). 13. Tsuge, T., Fukui, T., Matsusaki, H., Taguchi, S., Kobayashi, G., Ishizaki, A., and Doi, Y.: Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas ueruginosa and their use for polyhydroxyalkanoate
synthesis. FEMS Microbial. Lett., 184, 193-198 (2000). 14. Matsumoto, K., Nakae, S., Taguchi, K., Matsusaki, H., Seki, M., and Doi, Y.: Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) copolymer from sugars by recombinant Rulstoniu eutrophu harboring the phuCI,, and the phuG, genes of Pseudomonas sp. 61-3. Biomacromolecules, 2, 934-939 (2001). 15. Kichise, T., Taguchi, S., and Doi, Y.: Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromenus cuviae PHA synthase. Appl. Environ. Microbial., 68, 2411-2419 (2002). 16. Taguchi, S., Nakamura, H., Hiraishi, T., Yamato, I., and Doi, Y.: In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis. J. Biochem. (Tokyo), 131,801-806 (2002). 17. Modis, Y. and Wierenga, R. K.: Crystallographic analysis of the reaction pathway of Zoogloeu rumigera biosynthetic thiolase. J. Mol. Biol., 297, 1171-l 182 (2000). 18. Hisano, T., Fukui, T., Iwata, T., and Doi, Y.: Crystallization and preliminary X-ray analysis of (R)-specific enoyl-CoA hydratase from Aeromonus cuviae involved in polyhydroxy-
584
J. BIOSCI.BIOENG.,
TSUGE
alkanoate biosynthesis. Acta Crystallogr. D. Biol. Crystallogr., 57, 145-147 (2001). 19. Cboi, J. I., Lee, S. Y., and Han, K.: Cloning of the AZcaZigenes labs polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of poly(3-hydroxybutyrate) in Escherichia coli. Appl. Environ. Microbial., 64, 48974903 (1998). 20. Choi, J. I. and Lee, S. Y.: High-level production of poly(3hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli. Avvl. Environ. Microbial..
27.
28.
1.
65,4363-4368 (1999). 21. Lee, S.Y., Choi, J., and Wong, H.H.: Recent advances in polyhydroxyalkanoate production by bacterial fermentation. Int. J. Biol. Macromol., 25,31-36 (1999). 22. Yamane, T.: Cultivation engineering of microbial bioplastics production. FEMS Microbial. Rev., 103,257-264 (1992). 23. Chen, G. Q., Zhang, G., Park, S. J., and Lee, S. Y.: Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Appl. Microbial. Biotechnol., 57, 50-55 (2001). 24. Tsuge, T., Tanaka, K., and Ishizaki, A.: Development of a novel method for feeding a mixture of L-lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly-D-3hydroxybutyrate production. J. Biosci. Bioeng., 91, 545-550 (2001). 25. Tsuge, T,, Tanaka, K., Shimoda, M., and Ishizaki, A.: Optimization of L-lactic acid feeding for production of poly-D-3hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture. J. Biosci. Bioeng., 88,404-409 (1999). 26. Kohayashi, G., Tanaka, K., Itoh, I-I., Tsuge, T., Sonomoto,
29.
30.
3 1.
32.
K., and Ishizaki, A.: Fermentative production of P(3HBco3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method. Biotechnol. Lett., 22, 1067-1069 (2000). Miyake, M., Erata, M., and Asada, Y.: A thermophilic cyanobacterium, Synechococcus sp. MA19, capable of accumulating poly+hydroxybutyrate. J. Ferment. Bioeng., 82, 512-514 (1996). Nishioka, M., Nakai, K., Miyake, M., Asada, Y., and Taya, M.: Production of the poly-j3-hydroxyalkanoate by thermophilic cyanobacterium, S’echococcus sp. MA19, under phosphate-limitted condition. Biotechnol. Lett., 23, 1095-1099 (2001). Ishizaki, A., Tanaka, K., and Taga, N.: Microbial production of poly-D-3-hydroxybutyrate from CO,. Appl. Microbial. Biotechnol., 57,6-12 (2001). Ishizaki, A. and Tanaka, K.: Production of poly+hydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC176977 J. Ferment. Bioeng., 71,254-257 (1991). Sugimoto, T., Tsuge, T., Tanaka, K., and Ishizaki, A.: Control of acetic acid concentration by pH-stat continuous substrate feeding in heterotrophic culture phase of two-stage cultivation of Alcaligenes eutrophus for production of P(3HB) from CO,, H,, and 0, under non-explosive conditions. Biotechnol. Bioeng., 62,625-631 (1999). Tanaka, K., Ishizaki, A., Kanamaru, T., and Kawano, T.: Production of poly(D-3-hydroxybutyrate) from CO,, H,, and 0, by high cell density autotrophic cultivation of Alcaligenes eutrophus. Biotechnol. Bioeng., 45,268-275 (1995).