Abstract
Medium-chain-length-polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters containing monomers ranging from 6 to 14 carbons in length. The key enzymes of their biosynthesis are PHA-polymerase (product of phaC gene) and 3-hydroxyacyl-acyl carrier protein-CoA transferase (product of phaG gene). With aadA (aminoglycoside 3′-adenylyltransferase) gene as screening marker, two chloroplast transformation vectors of pTC2 harboring phaC2 gene only and pTGC harboring both phaC and phaG genes were constructed and introduced into tobacco chloroplast genome through particle bombardment. PCR and Southern blot analysis confirmed the insertion of the introduced genes into chloroplast genome. The content of mcl-PHAs accumulated in transgenic plants was analyzed by gas chromatography, mcl-PHAs accumulated up to 4.8 mg/g dry weight (dw) in transgenic line S4-3; their monomers were 3-hydroxyoctanoate and 3-hydroxydecanoate. Accumulation of mcl-PHAs polymers in the tobacco chloroplast was also observed by transmission electron microscopy. To our knowledge, this is the first report on the synthesis of mcl-PHAs in tobacco via chloroplast genetic engineering.
Similar content being viewed by others
References
Lee, S. Y., Bacterial polyhydroxyalkanoates, Biotec. Bioeng., 1996, 49: 1–14.
Anderson, A. J., Dawes, E. A., Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates, Microbiol. Rev., 1990, 54: 450–472.
Steinbüchel, A., Schlegel, H. G., Physiology and molecular genetics of poly (β-hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus, Mol. Microbiol., 1991, 5: 535–542.
Steinbüchel, A., Füchtenbusch, B., Bacterial and other biological systems for polyester production, Trends Biotechnol., 1998, 16: 419–427.
Poirier, Y., Dennis, D. E., Klomparens, K. et al., Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants, Science, 1992, 256: 520–523.
Nawrath, C., Poirier, Y., Somerville, C., Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation, Proc. Natl. Acad. Sci. USA, 1994, 91: 12760–12764.
Mittendorf, V., Robertson, E. J., Leech, R. M. et al., Synthesis of medium-chain-length polyhyroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid β-oxidation, Proc. Natl. Acad. Sci. USA, 1998, 95: 13397–13402.
John, M., Keller, G., Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells, Proc. Natl. Acad. Sci. USA, 1996, 93: 12768–12773.
Hahn, J. J., Eschenlauer, A. C., Sleytr, U. B. et al., Peroxisomes as sites for synthesis of polyhydroxyalkanoates in transgenic plants, Biotechnol. Prog., 1999, 15: 1053–1057.
Houmiel, K. L., Slater, S., Broyles, D. et al., Poly (β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus, Planta, 1999, 209: 547–550.
Nakashita, H., Arai, Y., Yoshioka, K. et al., Production of biodegradable polyester by a transgenic tobacco, Biosci. Biotech. Biochem., 1999, 63: 870–874.
Nakashita, H., Arai, Y., Shikanai, T. et al., Introduction of bacterial metabolism into higher plants by polycistronic transgene expression, Biosci. Biotechnol. Biochem., 2001, 65: 1688–1691.
Arai, Y., Nakashita, H., Doi, Y. et al., The plastid targeting of polyhydroxybutyrate biosynthetic pathway in tobacco, Plant Biotechnol., 2001, 18: 289–293.
Arai, Y., Shikanai, T., Doi, Y. et al., Production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid, Plant Cell Physiol., 2004, 45(9): 1176–1184.
Zhang, J. Y., Su, N., Zhang, Z. L. et al., Expression poly-3-hydroxybutyrate synthetic genes through chloroplast genetic engineering, Chinese Science Bulletin, 2002, 47(11): 845–848.
Romano, A., Raemakers, K., Visser, R. et al., Transformation of potato (Solarium tuberosum) using particle bombardment, Plant Cell Rep., 2001, 20: 198–204.
Romano, A., Raemakers, K., Bernardi, J. et al., Transgene organization in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes, Transgenic Res., 2003, 12: 461–473.
Romano, A., Vreugdenhil, D., Jamar, D. et al., Evidence of medium-chain-length polyhydroxyoctanoate accumulation in transgenic potato lines expressing the Pseudomonas oleovorans Pha-C 1 polymerase in the cytoplasm, Biochem. Eng. J., 2003, 16: 135–143.
Wróbel, M., Zebrowski, J., Szopa, J., Polyhydroxybutyrate synthesis in transgenic flax, Journal of Biotechnology, 2004, 107: 41–45.
Menzel, G., Harloff, H. J., Jung, C., Expression of bacterial poly (3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.), Appl. Micro. Bioech., 2003, 60: 571–576.
LÖssl, A., Eibl, C., Harloff, J. et al., Polyester synthesis in transplastomic tobacco (Nicotiana tabacum L.): Significant contents of polyhydroxybutyrate are associated with growth reduction, Plant Cell. Rep., 2003, 21: 891–899.
Romano, A., van der Plas, L. H., Witholt, B. et al., Expression of poly-3-Hydrozyalkanoate (PHA) polymerase and acyl-CoAtransacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs, Planta, 2005, 220(3): 455–464.
Gong, X. S., Yan, L. F., The method of isolation chloroplast DNA from higher plants, Chinese Science Bulletin, 1991, 36(6): 467–469.
Hiratsuka, J., Shimada, H., Whittier, R. et al., The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals, Mol. Gen. Genet, 1989, 217: 185–194.
Shinozaki, K., Ohme, M., Tanaka, M. et al., The complete nucleotide sequence of the tobacco chloroplast genome: Its gene organization and expression, EMBO J., 1986, 5: 2043–2049.
Wakasugi, T., Sugita, M., Tsudzuki, T. et al., Updated gene map of tobacco chloroplast DNA, Plant Mol. Biol. Rep., 1998, 16: 231–241.
Svab, Z., Maliga, P., High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene, Proc. Natl. Acad. Sci. USA, 1993, 90: 913–917.
Lin, J. A., An, J. L., Hong, J., Applied biological electron microscopy technique, Liao Ning Scientific Publishing Company, 1998, 48–52.
Huijberts, G., Eggink, G., Waard, de P. et al., Pseudomonas putida KT2442 cultivated on glucose accumulates poly (3-hydroxyalkanoates) consisting of saturated and unsaturated monomers, Appl. Environ. Microbiol., 1992, 58: 536–544.
Rehm, B., Kruger, N., Steinbuchel, A., A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis, J. Biol. Chem., 1998, 273: 24044–24051.
Author information
Authors and Affiliations
Corresponding authors
About this article
Cite this article
Wang, Y., Wu, Z., Zhang, X. et al. Synthesis of medium-chain-length-polyhydroxyalkanoates in tobacco via chloroplast genetic engineering. Chin.Sci.Bull. 50, 1113–1120 (2005). https://doi.org/10.1360/982005-130
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1360/982005-130