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

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.