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Synthesis of Short-Chain-Length/Medium-Chain Length Polyhydroxyalkanoate (PHA) Copolymers in Peroxisomes of Transgenic Sugarcane Plants

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Abstract

Metabolic engineering of crops is a potential route to economically viable production of polyhydroxyalkanoates (PHAs), biodegradable and renewable alternatives to conventional plastics. In particular, short-chain-length (SCL)/medium-chain-length (MCL) PHA copolymers have attracted commercial interest for their wide range of potential applications. To date, examples of SCL/MCL PHA copolymer production in plant peroxisomes have involved single transgene approaches in transgenic Arabidopsis. We attempted to produce SCL/MCL PHA copolymers using a multigene strategy in peroxisomes of the high biomass food and industrial crop, sugarcane (Saccharum hybrids). Our approach involved peroxisomal targeting of a 3-ketothiolase, acetoacetyl-CoA reductase, enoyl-CoA hydratase and PHA synthase, as well as plastid targeting of a acyl-ACP thioesterase and 3-ketoacyl-ACP synthase to increase peroxisomal β-oxidation flux. Of 143 transgenic sugarcane lines generated by co-bombardment with the six transgenes, six were identified with PHA copolymers at up to 0.015% leaf dry mass, consisting mainly of saturated C4–C16 3-hydroxyalkanoic acids. One line with high acetoacetyl-CoA reductase and low 3-ketothiolase transcript levels had increased 3-hydroxybutyrate content, and acyl-ACP thioesterase and 3-ketoacyl-ACP synthase expression were associated with altered MCL monomer profiles. SCL/MCL PHA copolymer from the highest-yielding line showed a weight-average molecular weight of 111 KDa and polydispersity index of 1.2. Transmission electron microscopy of leaf sections from this line indicated the presence of PHA granules in peroxisomes. This work demonstrates SCL/MCL PHA copolymer biosynthesis in sugarcane peroxisomes and provides a basis for further development of mechanisms for controlling PHA composition in transgenic crop plants.

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Abbreviations

ACP:

Acyl carrier protein

DM:

Dry mass

HPLC:

High performance liquid chromatography

PDI:

Polydispersity index

PHA:

polyhydroxyalkanoate

PHB:

polyhydroxybutyrate

SCL:

short-chain-length

MCL:

medium-chain-length

GC-MS:

gas chromatography–mass spectrometry

GPC:

gel permeation chromatography

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Acknowledgements

This work was funded jointly by the Australian Government through the Co-operative Research Centre for Sugarcane Industry Innovation through Biotechnology (CRCSIIB) and BSES Limited. AG was a recipient of a Smart State Fellowship awarded by the Department of State Development, Trade and Innovation of the Queensland Government. We wish to thank Ms. Liz Burns (BSES Limited) for assistance with initial GC-MS screening; Mr. Niall Masel, BSES Limited, for assistance with GPC analysis; Dr. Deb Stenzel, Queensland University of Technology, and Ms. Kimberley Tilbrook, The University of Queensland, for assistance with transmission electron microscopy.

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  1. Stevens M. Brumbley

    Present address: Department of Biological Science, The University of North Texas, 1155 Union Circle, # 305220, Denton, TX, 76203-5017, USA

  2. David J. Anderson

    Present address: School of Agricultural and Food Sciences, The University of Queensland, Brisbane, QLD, 4072, Australia

  3. Annathurai Gnanasambandam

    Present address: Department of Primary Industries, Horsham, VIC, 3400, Australia

Authors and Affiliations

  1. BSES Limited, 50 Meiers Road, Indooroopilly, QLD, 4068, Australia

    David J. Anderson, Annathurai Gnanasambandam, Edwina Mills, Michael G. O’Shea & Stevens M. Brumbley

  2. Cooperative Research Centre for Sugar Industry Innovation through Biotechnology, The University of Queensland, Brisbane, QLD, 4072, Australia

    David J. Anderson, Annathurai Gnanasambandam, Edwina Mills, Michael G. O’Shea, Lars K. Nielsen & Stevens M. Brumbley

  3. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, 4072, Australia

    Lars K. Nielsen & Stevens M. Brumbley

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  1. David J. Anderson
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Correspondence to Stevens M. Brumbley.

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Communicated by: Robert Birch

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Anderson, D.J., Gnanasambandam, A., Mills, E. et al. Synthesis of Short-Chain-Length/Medium-Chain Length Polyhydroxyalkanoate (PHA) Copolymers in Peroxisomes of Transgenic Sugarcane Plants. Tropical Plant Biol. 4, 170–184 (2011). https://doi.org/10.1007/s12042-011-9080-7

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