Abstract
In order to improve the production of poly(3-hydroxybutyrate) [P(3HB)], two different recombinant bacteria, Escherichia coli and Alcaligenes eutrophus were investigated. Firstly, recombinant E. coli harboring a stable high copy number plasmid pSYL107 containing the A. eutrophus polyhydroxyalkanoate (PHA) synthesis genes and E. coli ftsZ gene was employed for the production of P(3HB) by fed-batch culture in a defined medium. Suppression of filamentation by overexpressing the cell division protein FtsZ allowed the production of P(3HB) to a high concentration of 104 g/L, and P(3HB) content of 70% with high productivity of 2 g P(3HB)/L-h in a defined medium, which was the highest value reported to date by employing recombinant E. coli. Secondly, wild P(3HB) producer A. eutrophus was metabolically engineered to amplify the activities of the three enzymes, PHA synthase, beta-ketothiolase, and NADPH-dependent reductase, involved in the synthesis of P(3HB). The A. eutrophus PHA synthesis genes were cloned into a broad-host-range plasmid pVK101 and the constructed vector system was transferred into A. eutrophus by electroporation. In flask culture, the final cell concentration of recombinant A. eutrophus increased with decreasing carbon/nitrogen (C/N) molar ratio. On the other hand, P(3HB) concentration was highest at the medium C/N molar ratio. For the fixed C/N molar ratio, the concentrations of cell and P(3HB) were increased with increasing glucose concentration. Comparison of cell growth and P(3HB) production by recombinant and wild type A. eutrophus in batch cultures showed that the final P(3HB) concentration, P(3HB) content, P(3HB) synthesis rate were all higher in the recombinant strain compared with the wild type.
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References
Anderson, A. J., and Dawes, E. A., 1990, Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54:450.
Brandi, H., Gross, R. A., Lenz, R. W., and Fuller, R. C., 1990, Plastics from bacteria and for bacteria: poly(β-hydroxyalkanoates) as natural, biocompatible, and biodegradable polyesters. Adv. Biochem. Eng. Biotechnol. 41:77.
Braunegg, G., Sonnleitner, B., and Lafferty, R. M, 1978, A rapid gas Chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Eur. J. Appl. Microbiol. Biotechnol. 6:29.
Byrom, D., 1987, Polymer synthesis by microorganisms: technology and economics. Trends Biotechnol. 5:246.
Byrom, D., 1991, Biomaterials: novel materials from biological sources, Stockton, NY.
Byrom, D., 1994, Polyhydroxyalkanoates, pp. 5-33. in: Plastics from microbes: microbial synthesis of polymers and polymer precursors. D. P. Mobley, ed., Hanser Munich.
Doi, Y., 1990, Microbial polyesters, VCH, NY.
Dower, W. J., Miller, J. F., and Ragsdale, C. W., 1988, High efficiency transformation of Escherichia coli by high voltage electroporation. Nucleic Acids Res. 16:6127.
Fidler, S., and Dennis, D., 1992, Polyhydroxyalkanoate production in recombinant Escherichia coli. FEMS Microbiol Rev. 103:231.
Friehs, K., and Lafferty, R. M., 1989, Cloning and expression of the levanase gene in Alcaligenes eutrophus H16 enables the strain to hydrolyze sucrose. J. Biotechnol. 10:285.
Haywood, G. W., Anderson, A. J., Chu, L., and Dawes, E. A., 1988, The role of NADH-and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxyalkanoate synthesizing organism Alcaligenes eutrophus. FEMS Microbiol. Lett. 52:259.
Holmes, P. A., 1985, Applications of PHB-A microbially produced biodegradable thermoplastic. Phys. Technol. 16:32.
Holmes, P. A., 1988, Biologically produced PHA polymers and copolymers, p. 1–65. in: Developments in crystalline polymers, D. C. Bassett, ed., Vol. 2. Elsevier, London.
Kim, B. S., Lee, S. C., Lee, S. Y., Chang, H. N., Chang, Y. K., and Woo, S. I., 1994a, Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnol. Bioeng. 43:892.
Kim, B. S., Lee, S. C., Lee, S. Y., Chang, H. N., Chang, Y. K., and Woo, S. I., 1994b, Production of poly-(3-hydroxybutyric-co-3-hydroxyvaleric acid) by fed-batch culture of Alcaligenes eutrophus with substrate feeding using on-line glucose analyzer. Enzyme Mi crobial Technol. 16:556.
Kim, B. S., Lee, S. Y., and Chang, H. N., 1992, Production of poly-β-hydroxybutyrate by fed-batch culture of recombinant Escherichia coli. Biotechnol. Lett. 14:811.
Knauf, V. C., and Nester, E. W., 1982, Wide host range cloning vectors: A cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid 8:45.
Lafferty, R. M., Korsatko, B., and Korsatko, W., 1988, Microbial production of poly-β-hydroxybutyric acid, pp. 135–176. in: Biotechnology, H. J. Rehm and G. Reed, eds., vol. 6b. VCH, Weinheim.
Lee, I. Y., Kim, M. K., Park, Y. H., and Lee, S. Y., 1996, Regulatory effects of cellular nicotiamide nucleotides and enzyme activities on poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli. Biotechnol. Bioeng, 52:707.
Lee, S. Y., 1994, Suppression of filamentation in recombinant Escherichia coli by amplified FtsZ activity. Biotechnol. Lett. 16:1247.
Lee, S. Y., 1996a, Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49:1.
Lee, S. Y., 1996b, Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria. Trends Biotechnol. 14:431.
Lee, S. Y., and Chang, H. N., 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.
Lee, S. Y., and Chang, H. N., 1995a, Production of poly-(hydroxyalkanoic acid). Adv. Biochem. Eng. Biotechnol. 52:27.
Lee, S. Y., and Chang, H. N., 1995b, Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studied. Can. J. Microbiol. 41(Suppl. 1): 207.
Lee, S. Y., Chang, H. N., and Chang, Y. K., 1994a, Production of poly(β-hydroxybutyric acid) by recombinant Escherichia coli. Ann. NY Acad. Sci. 721:43.
Lee, S. Y., Lee, K. M., Chang, H. N., and Steinbuchel, A., 1994b, Comparison of Eschericia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid), and morphological changes. Biotechnol. Bioeng. 44:1337.
Lee, S. Y., Yim, K. S., Chang, H. N., and Chang, Y. K., 1994c, Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) in Escherichia coli. J. Biotechnol. 32:203.
Lowry, O. H., Rosenbrough, N. S., Farr, A.L., and Pandall, R. T., 1951, Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265.
Nishimura, T., Sato, T., and Tomita, K., 1978, Purification and properties of β-ketothiolase from Zoogloea ramigera. Arch. Microbiol. 116:21.
Peoples, O. P., and Sinskey, A. J., 1989, Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). J. Biol Chem. 264:15298.
Pries, A., Steinbuchel, A., and Schlegel, H. G., 1990, Lactose-, and galactose-utilizing strains of poly(hydroxyalkanoic acid)-accumulating Alcaligenes eutrophus and Pseudomonas saccharophila obtained by recombinant DNA technology. App. Microbiol. Biotechnol. 33:410.
Sambrook, J., Fritsch, E. F., and Maniatis, T., 1989, Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, NY.
Schubert, P., Steinbuchel, A., and Schlegel, H. G., 1988, Cloning of the Alcaligenes eutrophus poly-β-hydroxybutyrate synthetic pathway and synthesis of PHB in Escherichia coli. J. Bacteriol. 170:5837.
Slater, S. C., Voige, W. H., and Dennis, D. E., 1988, Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-β-hydroxybutyrate biosynthetic pathway. J. Bacteriol 170:4431.
Steinbuchel, A., 1991, Polyhydroxyalkanoic acids, p. 124–213. in Biomaterials: novel materials from biological sources, D. Byrom, ed., Stockton, New York.
Yamane, T., Fukunaga, M., and Lee, Y. W., 1996, Increased PHB productivity by high-cell-density fed-batch culture of Alcaligenes latus, a growth-associated PHB producer. Biotechnol. Bioeng. 50:197.
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Lee, S.Y., Choi, Ji., Wang, F. (1998). Production of Poly(3-Hydroxybutyrate) by Recombinant Bacteria. In: Prasad, P.N., Mark, J.E., Kandil, S.H., Kafafi, Z.H. (eds) Science and Technology of Polymers and Advanced Materials. Springer, Boston, MA. https://doi.org/10.1007/978-1-4899-0112-5_40
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