ISOLATION AND UTILIZATION OF ACETYL-CoA CARBOXYLASE FROM OIL PALM (Elaeis guineensis) MESOCARP

One of the targets of the MPOB oil palm genetic engineering programme is to synthesize biodegradable plastics. Biodegradable plastics were first discovered in bacterial systems. Polyhydroxybutyrate (PHB), the most common biodegradable plastic, is synthesized from acetyl-CoA by the sequential action of the following three enzymes: beta-ketothiolase (phbA or bktB), acetoacetyl-CoA reductase (phbB) and PHB synthase (phbC). Acetyl-CoA is also the main substrate fir fatty acid synthesis, where acetyl-CoA carboxylase (ACCase)catalyses the conversion of acetyl-CoA to malonyl-CoA, the building block for fatty acid synthesis. Down-regulating ACCase could divert the central metabolite acetyl-CoA to higher value products such as PHB. in this study, efforts were made to isolate both the multifunctional form of ACCase and biotin carboxylase (BC), a component protein of the multisubunit form of ACCase. Initially, reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers designed based on the conserved region of plant biotin carboxylase gene was used to amplify a partial length of the oil palm cDNA. This was then used for further isolation of the full length cDNA by random amplification of cDNA ends (RACE), followed by end-to-end PCR. The RT-PCR was similary used to isolate a partial length multifunctional ACCase employing degenerate primers designed based on conserved regions of plant ACCase. After confirmation through sequencing and cross-reference with gene bank, the partial length cDNA of multifunctional ACCase was incorporated in an intervention strategy, where the cDNA was added in the antisense orientation into existing PHB and PHBV transformation vectors driven by an oil palm mesocarp specific (MSP1) promoter. It was envisaged that by down-regulating the activity of ACCase, fatty acid biosynthesis activity will be reduced and thus the acetyl-CoA pool diverted to production of PHB and PHBV The resulting vectors were later transformed into oil palm embryogenic calli using the Biolistics (TM) approach. After selection on medium containing the herbicide Basta, resistant colonies were isolated and are currently undergoing regeneration into full plants.