Journal of Biotechnology 147 (2010) 31–36 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Design of a single-chain multi-enzyme fusion protein establishing the polyhydroxybutyrate biosynthesis pathway Jane A. Mullaney, Bernd H.A. Rehm ∗ Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand article info Article history: Received 9 September 2009 Received in revised form 23 February 2010 Accepted 26 February 2010 Keywords: Biopolyester Polyhydroxyalkanoate Fusion protein Biopolymer abstract Polyhydroxyalkanoates are biodegradable biocompatible polymers naturally produced by various bac- teria and archaea. Biotechnological production in transgenic plants has already been demonstrated with efficient polyhydroxybutyrate production requiring targeting of the enzymes to the chloroplasts. Three enzymes are required to establish the polyhydroxybutyrate biosynthesis pathway in non-naturally pro- ducing microorganisms or plants. To facilitate production of biopolyesters in plants, a gene encoding a translational fusion of the polyhydroxybutyrate biosynthesis enzymes PhaA (-ketothiolase), PhaB (acetoacetyl-CoA reductase) and PhaC (PHA synthase) was constructed. Escherichia coli harboring a plasmid encoding this fusion protein (PhaA–PhaB–PhaC) under control of the lac promoter accumu- lated polyhydroxybutyrate contributing to 0.4% (w/w) of cellular dry weight. Insertion of an extended linker between PhaA and PhaB increased polyhydroxybutyrate accumulation to 3.9% (w/w) of cellular dry weight. Introduction of a second plasmid encoding PhaA and PhaB restored polyhydroxybutyrate accumulation to wildtype levels of about 35% (w/w) of cellular dry weight suggesting that the func- tions of PhaA and/or PhaB were limiting factors. Deletion of PhaA in trans led to significantly reduced polyhydroxybutyrate production suggesting that the PhaA activity in the fusion protein is reduced. This study showed that a single-chain translational fusion protein comprising the three enzymes essential for polyhydroxybutyrate synthesis can be engineered which will strongly facilitate the establishment of recombinant polyhydroxybutyrate production organisms particularly requiring targeting to sub-cellular compartments such as the chloroplasts in plants. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Polyhydroxyalkanoates (PHAs) are biodegradable biocompati- ble polymers that serve as carbon and energy storage material and are deposited as spherical inclusions in the cytoplasm of bacteria and archaea. PHAs have been considered as renewable alterna- tive polyesters showing material properties suitable to replace oil-based thermoplastics (Chen et al., 2001; Lee et al., 1999; Rehm and Steinbüchel, 1999). Polyhydroxybutyrate (PHB) is the most common PHA produced by various bacteria and archaea. Biosynthesis of PHB requires the activity of three enzymes, with the genes encoding these PHB biosynthesis proteins often co- localized and organized in an operon (Rehm and Steinbüchel, 1999). PHB biosynthesis enzymes are (i) the -ketothiolase (PhaA), which catalyses condensation of the central metabolite acetyl- CoA (acetyl-Coenzyme A) to acetoacetyl-CoA, (ii) the (R)-specific acetoacetyl-CoA reductase (PhaB), which catalyses stereo-specific reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA and (iii) ∗ Corresponding author. Tel.: +64 6 350 5515x7890; fax: +64 6 350 2267. E-mail address: b.rehm@massey.ac.nz (B.H.A. Rehm). the polyester synthase (PhaC) which polymerizes this substrate to poly-(R)-3-hydroxybutyrate (Rehm, 2003, 2006). Industrial production of PHA was first obtained by using genetically engineered Escherichia coli harboring all three biosyn- thesis genes from Cupriavidus necator (for review see Rehm and Steinbüchel, 1999). More recently the three enzyme based biosyn- thesis pathway was established in plants (Poirier et al., 1992; Snell and Peoples, 2002). PHB/PHA production in plants has been con- sidered because of the increased plant biomass when compared to bacterial mass used for PHB production. Unlike bacteria, the plants do not require sterile conditions and production costs are proposed to be lower as plants can obtain their carbon directly from car- bon dioxide (CO 2 )(Poirier et al., 1992; Suriyamongkol et al., 2007). Transfer of genes encoding the PHB synthesis enzymes into plants has led to PHB production in Arabidopsis (Nawrath et al., 1994), cotton (John and Keller, 1996), switchgrass (Somleva et al., 2008), tobacco (Lössl et al., 2003), rapeseed (Houmiel et al., 1999), corn (Poirier and Gruys, 2002), flax (Wrobel et al., 2004), and sugar- cane (Petrasovits et al., 2007). However for efficient production, enzymes had to be targeted to the chloroplasts (Houmiel et al., 1999; Lössl et al., 2003; Nawrath et al., 1994; Petrasovits et al., 2007; Poirier and Gruys, 2002; Wrobel et al., 2004) and constitu- 0168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.02.021