title: The use of an acetoacetyl-CoA synthase in place of a β-ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells.
abstract: Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing β-ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the β-ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10(6)  Da. These results are a major step forward in engineering a high biomass C4 grass for the commercial production of PHB.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, Qld, Australia.
authors.forename: Richard B
authors.initials: RB
authors.lastname: McQualter
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, Qld, Australia.
authors.forename: Lars A
authors.initials: LA
authors.lastname: Petrasovits
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, Qld, Australia.
authors.forename: Leigh K
authors.initials: LK
authors.lastname: Gebbie
authors.affiliation: Metabolix Inc., Cambridge, MA, USA.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schweitzer
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, Qld, Australia.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Blackman
authors.affiliation: Metabolomics Australia Queensland Node, Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, Qld, Australia.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Chrysanthopoulos
authors.affiliation: Central Analytical Research Facility, Queensland University of Technology, Brisbane, Qld, Australia.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Hodson
authors.affiliation: Central Analytical Research Facility, Queensland University of Technology, Brisbane, Qld, Australia.
authors.forename: Manuel R
authors.initials: MR
authors.lastname: Plan
authors.affiliation: Metabolomics Australia Queensland Node, Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, Qld, Australia.
authors.forename: James D
authors.initials: JD
authors.lastname: Riches
authors.affiliation: Metabolix Inc., Cambridge, MA, USA.
authors.forename: Kristi D
authors.initials: KD
authors.lastname: Snell
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, Qld, Australia.
authors.forename: Stevens M
authors.initials: SM
authors.lastname: Brumbley
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, Qld, Australia.
authors.forename: Lars K
authors.initials: LK
authors.lastname: Nielsen
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D018533:Biomass; D053898:Biosynthetic Pathways; D002736:Chloroplasts / Q000235:genetics; D018556:Crops, Agricultural; D006885:Hydroxybutyrates / Q000378:metabolism*; D058503:Mesophyll Cells / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D018087:Plastids / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism*; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D031786:Saccharum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000094122:Polyhydroxybutyrates
chemical_list: D000102:Acetyl-CoA C-Acyltransferase; D000214:Acyl Coenzyme A; D006885:Hydroxybutyrates; D010940:Plant Proteins; D011091:Polyesters; D054813:Polyhydroxyalkanoates; C010667:acetoacetyl CoA; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates
keywords: NphT7; PHB; acetoacetyl-CoA synthase; biopolymer; polyhydroxybutyrate; sugarcane; β-ketothiolase