gene isolation – Journal of Oil Palm Research

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

mpob_admin — Sat, 13 Nov 2021 15:23:25 +0000

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: β-ketothiolase (phbA or bktB), acetoacetyl-CoA reductase (phbB) and PHB synthase (phbC). Acetyl-CoA is also the main substrate for 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 wasadded 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 BiolisticsTM approach. After selection on medium containing the herbicide Basta, resistant colonies were isolated and are currently undergoing regeneration into full plants.

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

mpob_admin — Sat, 13 Nov 2021 15:23:25 +0000

The enzyme β-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 β-ketothiolase activity in oil palm mesocarp crude extracts was designed and optimized. The highest levels of β-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.) β-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 β-ketothiolase cDNA encodes a cytosolic protein.
Genomic DNA gel blot analysis suggested a small family of β-ketothiolase isogenes. Northern analysis revealed that β-ketothiolase mRNA 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 β-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 β-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.

THE ISOLATION AND CHARACTERISATION OF OIL PALM (Elaeis guineensis Jacq.) β-KETOACY-ACYL CARRIER PROTEIN (ACP) SYNTHASE (KAS) II cDNA

mpob_admin — Sat, 13 Nov 2021 08:04:16 +0000

Modulating endogenous levels and/or producing novel fatty acids of oils have gained significant attention in recent years to meet the demand for oils for specific markets. The commodity palm oil is composed mainly of four fatty acids: palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1) and linoleic acid (18:2). The percentages of these fatty acids in palm oil average 44%, 4%, 39% and 10%, respectively, with trace amounts of other fatty acids. Metabolic engineering may be used to produce oil crops with desired fatty acid compositions. We have isolated and characterised β-ketoacyl ACP-synthase II (KASII) cDNA from oil palm (Elaeis guineensis) which is one of the main components for the oil palm genetic engineering programme. KAS II is associated with the accumulation of palmitic acid in oil palm, and its role in controlling the ratio of C16:C18 has been previously determined. We isolated KAS II cDNA from oil palm, and functionally characterised the same in Escherichia coli and Arabidopsis. Partial length KAS II cDNA was first obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR). Rapid amplification of cDNA ends (RACE) was then used to isolate both the 5’ and 3’ ends of the KAS II sequences. Assembly of the partial length sequence fragments, including the 5’ and 3’ ends, allowed for the full-length sequence information on the KAS II cDNA to be obtained and used in the gene isolation. Expression studies in E. coli resulted in an increase in oleic acid at the expense of palmitic acid. Arabidopsis thaliana was also used to further confirm the functional activity of the oil palm KAS II. A significant decrease in C18:0 and accumulation of C16:0 were detected in the plants that had been transformed with the antisense KASII construct. This suggests that the substrate specificity of the oil palm KAS II is similar to that of KAS II from other plants which preferentially elongate palmitic to stearic acids. The oil palm KAS II may, therefore, be useful in providing new opportunities in the genetic engineering programme for the production of high-value products such as an oil with a high content of monounsaturated fatty acids from the transgenic oil crops.