ACTIVITY STUDIES, GENE CHARACTERIZATION AND MANIPULATION OF β-KETOTHIOLASE OF OIL PALM (Elaeis guineensis Jacq.) MESOCARP

The enzyme beta-ketothiolase plays a major role in isoprenoid metabolism as well as in polyhydroxybutyrate (PHB) biosynthesis. PHB is a member of the polyhydroxyalkanoate (PHA), family of polymers with potential as biodegradable replacements for the current petrochemical plastics used. A coupled assay system for beta-ketothiolase activity in oil palm mesocarp crude extracts was designed and optimized. The highest levels of beta-ketothiolase specific activity were seen in oil palm fruits 8 to 11 weeks after anthesis (WAA) with ripe fruits showing lower activity. A cDNA coding for oil palm (Elaeis guineensis Jacq.) beta-ketothiolase was isolated through RT-PCR and RACE techniques. The longest reading frame encoded a protein of 415 amino acids with a predicted relative molecular weight of 43 217 Da, and considerable similarities to the gene/enzyme in other plant thiolases and, to a lesser extent, prokaryotic thiolases. There is no evidence for the presence of a signal peptide, suggesting that the beta-ketothiolase cDNA encodes a cytosolic protein. Genomic DNA gel blot analysis suggested a small family of beta-ketothiolase isogenes. Northern analysis revealed that beta-ketothiolase in RNA transcripts are present in higher quantities in the riper (13,17,20 WAA) than younger fruits at 6, 8 and 11 WAA, contradicting the biochemical activity profile. This discrepancy may be caused by interfering substances in the oil palm crude extract such as lipids or competition for substrates by other enzymes. Other explanatory factors include genotype dependency, thiolase mRNA transcripts of the same size and post-translational modification. The expression profile obtained in the Northern analysis is in agreement with that of sterol and carotenoid accumulation during fruit ripening. Sequence analysis with biocomputing tools showed that beta-ketothiolase cDNA is relatively lowly expressed in oil palm mesocarp throughout its development. The gene was used in an intervention strategy to substitute for bacterial beta-ketothiolase by redesigning the PHB transformation vector driven by oil palm mesocarp-specific promoter (MSP1), for synthesizing biodegradable plastics in oil palm. The new vector, designated pMS35, was later transformed into oil palm embryogenic calli using the biolistics approach. Currently, Basta-resistant embryogenic calli have been obtained and are undergoing proliferation and regeneration.