CONSTRUCTION OF PHB AND PHBV TRANSFORMATION VECTORS FOR BIOPLASTICS PRODUCTION IN OIL PALM 37 CONSTRUCTION OF PHB AND PHBV TRANSFORMATION VECTORS FOR BIOPLASTICS PRODUCTION IN OIL PALM ABDUL MASANI MAT YUNUS*; HO CHAI-LING** and GHULAM KADIR AHMAD PARVEEZ* Keywords: poly-3-hydroxybutyrate (PHB), polyhyroxybutyrate-co-valerate (PHBV), constitutive promoters, tissue-specific promoter, plastid, transformation vectors. Date received: 31 July 2007; Sent for revision: 17 August 2007; Received in final form: 25 October 2007; Accepted: 7 November 2007. ABSTRACT The construction of transformation vectors carrying bioplastic biosynthetic genes driven by constitutive and oil palm mesocarp-specific promoters was completed. Four planned transformation vectors were produced. The poly-3-hydroxybutyrate (PHB) producing constructs carried the phbA, phbB and phbC genes, while the polyhyroxybutyrate-co-valerate (PHBV) producing constructs carried the bktB, phbB, phbC and tdcB genes. Each of these genes was fused with the transit peptide (Tp) of the oil palm acyl-carrier-protein (ACP) for targeting into the plastids of plant cells. All vectors carry the phosphinothricin acetyltransferase gene (bar) driven by an ubiquitin promoter as plant selectable marker. The matrix attachment region from tobacco (RB7MAR) was also included for stabilization of the transgene expression and to minimize the gene silencing due to positional effects. All constructs were verified by restriction analysis, polymerase chain reaction (PCR) and DNA sequencing. * Malaysian Palm Oil Board, P. O. Box 10620, 50720 Kuala Lumpur, Malaysia. E-mail: masani@mpob.gov.my ** Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. INTRODUCTION Poly-3-hydroxybutyrate (PHB) is the most common polyhydroxyalkanoate (PHA) produced as a storage material by some bacteria under restricted growth condition (Senior and Dawes, 1973). Biotechnological production of PHA was first made in the 1980s by Imperial Chemical Industries (ICI) using a polymer- accumulating bacterium, Ralstonia eutropha (Somerville et al., 1994). The cost of production was high because the most useful polymer, polyhyroxybutyrate-co-valerate (PHBV), could only be effectively synthesized by supplementing the fermentation medium with propionate. The commercially produced PHBV is known as Biopol TM . However, its commercial applications are limited due to the high production cost. Plants offer an alternative large-scale factory for the production of complex products. A variety of PHA having different physical properties are now being synthesized in a number of transgenic plants, including Arabidopsis (Poirier et al., 1992; Nawrath, et al., 1994; Bohmert et al., 2000), tobacco (Arai et al., 2001; Bohmert et al., 2002), rape (Houmiel et al., 1999), cotton (John and Keller, 1996), maize (Hahn et al., 1997) alfalfa (Saruul et al., 2002) and flax (Wrobel et al., 2004). These were accomplished by introducing bioplastic biosynthetic genes into the cytoplasm or specific compartments such as the plastids. The production of PHB in the cytoplasm was only 0.1% dry weight (dwt) (Poirier et al., 1992), approximately 900 times lower than PHB production in the bacterium R. eutropha. Furthermore, growth of the transgenic plants producing PHB was reduced compared to the untransformed plants. This may due to the limited supply of acetyl-CoA (Nawrath et al., 1994). The expression of PHB genes in plant compartments, such as the plastids, was thought to be a potential solution for PHB production in Journal of Oil Palm Research Special Issue on Malaysia-MIT Biotechnology Partnership Programme: Volume 2 - Oil Palm Metabolic Engineering (July 2008) p. 37-55