Category Archives: 2008 Special Issue July

Mesocarp-specific metallothionein-like gene promoter for genetic engineering of oil palm

Primers from within the coding region were used to capture the 5′ regulatory sequence of the mesocarpspecific metallothionein-like gene, MT3-A, via PCR-based genome walking. The amplified 1040 bp genomic fragment was cloned and sequenced. The sequence of the genomic clone showed total homology with the MT3-A cDNA sequence within their overlapping regions. Rapid amplification of 5’-cDNA ends (5’-RACE) was used to determine the full length cDNA sequence and the putative transcription site of the gene. The adenine residue at the 5’-end of the RACE product was chosen as the likely transcription start site. The 986 bp promoter region upstream of the adenine contains putative regulatory elements including a TATA box, an ethylene responsive element in reverse orientation and two I-boxes. Functional analysis of the MT3-A promoter was performed using a transient assay system. Transient expression of ß-glucuronidase (GUS) examined using qualitative histochemical GUS assay can be detected in both oil palm mesocarp and leaf tissue slices bombarded with the pBI221 transformation vector which contains the GUS reporter gene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. However, when the CaMV-35S promoter was replaced with MT3-A promoter in the transformation vector and used for bombardment, transient expression of GUS was detected in the oil palm mesocarp slices only and not in the leaf tissue. This suggests that the MT3-A promoter can be used to target specific gene expression into oil palm mesocarp tissues.

Isolation and high resolution microscopy of native bacterial polyhydroxybutyrate inclusions

Polyhydroxybutyrate (PHB) is a carbon reserve in some bacteria and under nutrient-limiting conditions, accumulates intracellularly in the form of inclusion bodies. These inclusions contain proteins, and the PHB within the inclusions exists in an amorphous state. In this study, a procedure to recover native PHB inclusions was developed, and the isolated inclusions characterized using 13C NMR, western blotting, atomic force microscopy and fluorescence microscopy. High resolution images of native PHB granules were obtained and the surface features and dimensions of the granules analysed. The size, morphology and composition of the PHB inclusions are important parameters for analysing inclusions formed within PHB-producing oil palm to support commercial objectives. The methods reported in this work support the development of analytical techniques for transgenic bioplastic-producing oil palm samples.

Light-harvesting chlorophyll a/b binding protein (LHCB) promoter for targeting specific expression in oil palm leaves

Oil palm leaves have the potential to be manipulated as a green factory to produce novel metabolites. In order to direct the specific expression of transgenes into the leaf tissue, a leaf-specific promoter is required. In these studies, efforts were carried out to isolate a leaf-specific gene and its regulatory sequence from the oil palm. Rapid amplification of 5’-cDNA Ends (5’-RACE) resulted in isolation of a 962 bp full length sequence of the light-harvesting chlorophyll a/b binding protein (LHCB). The amino acid sequence of this gene exhibited more than 88% homology with the LHCB1 gene isolated from monocots and dicots. Characterization of this transcript using Northern blot analysis revealed LHCB to be abundantly expressed in oil palm green leaf tissues, but not in the non-photosynthetic tissues such as kernel, mesocarp, germinated seedlings and inflorescences. Results from Southern blot analysis confirmed that at least four copies of the LHCB gene are present in the oil palm genome. The promoter region of LHCB was then obtained through genome walking approach. Based on the sequence analysis, it was found that the LHCB promoter lacks a TATA-box. Initiation of transcription can therefore be replaced by an initiator element (Inr) located at positions -1 to -7. Furthermore, a few putative cis-acting elements responsive to light, wounding, abscisic acid and heat-shock were also found in the distal and proximal regions of the LHCB promoter.

Non-radioactive assay for acetyl-CoA carboxylase activity

Acetyl-CoA carboxylase is a key enzyme in oil biosynthesis and is critical for the oil deposition pathway. This biotinylated enzyme catalyzes the first committed step in fatty acid biosynthesis, the ligation of a carbon to acetyl-CoA to form malonyl-CoA. The acetyl-CoA carboxylase holoenzyme has four distinct protein domains: biotin carboxylase, biotin carboxylase carrier protein, and the alpha and beta transcarboxylase domains. Biotin carboxylase catalyzes the addition of carbon dioxide to biotin carboxylase carrier protein, while the alpha and beta subunits of carboxyltransferase ligate the activated CO2 to acetyl-CoA. The canonical assays for monitoring the activity of acetyl-CoA carboxylase rely on incorporation of radiolabelled acetyl-CoA. In this work, we describe the development of a discontinuous, non-radioactive spectrophotometric assay for acetyl-CoA carboxylase activity. Permeabilized Corynebacterium glutamicum cells were added to an assay mixture containing acetyl-CoA, bicarbonate, magnesium and ATP, and aliquots were removed at set time points and stopped by the addition of trifluoroacetic acid. The level of acetyl-CoA remaining in each aliquot was determined via a citrate synthase assay, in which the formation of the yellow compound dithiobisnitrobenzoic acidthiophenolate was followed spectrophotometrically at 412 nm.

Construction of PHB and PHBV transformation vectors for bioplastics production in oil palm

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.

Reagents for generation and analysis of bioplastic producing plants

Polyhydroxyalkanoates (PHAs) are a class of biodegradable polyoxoesters synthesized from acetyl-CoA that naturally accumulate as intracellular granules in a diverse range of bacteria. Biosynthesis of the simplest PHA (PHB, poly-3-hydroxybutyrate) can be accomplished though the action of three enzymes, beta-ketothiolase, acetoacetyl-CoA reductase and PHA polymerase. We constructed plasmids that contain genetic elements for the production of PHB, or the closely related copolymer PHBV (poly-3-hydroxybutyrate-co-3-hydroxyvalerate), in oil palm, which produces abundant levels of acetyl-CoA. These bacterial PHA biosynthetic genes have been engineered to include plant plastid targeting signals, in order to direct biosynthesis of the polymer inside the plastids. In addition, we generated antibodies for the detection of PHA biosynthetic enzymes. The plasmids and antibodies reported in this work should be suitable as tools and reagents for the construction and analysis of PHA-producing oil palm.

Quantitative analysis of flux control over lipid biosynthesis in oil palm (Elaeis guineensis) mesocarp

Plant storage oils are of major commercial importance, yet our understanding of the regulation and control of their synthesis is poor. The control of lipid biosynthesis fluxes in the mesocarp of oil palm (Elaeis guineensis) was studied using modular (top-down) metabolic control analysis (TDCA). This allowed us to determine the relative contribution of two groups of reactions, fatty acid formation (Block A) and lipid assembly (Block B), to the control structure of overall pathway to triacylglycerol. The pathway was manipulated in two ways. Single manipulation involved the addition of oleate which inhibited fatty acid formation in Block A and stimulated lipid assembly in Block B. In double manipulation experiments, cerulenin was used in inhibition of Block A and Block B was inhibited by bromooctanoate. Single manipulation-TDCA revealed that the group flux control coefficient for fatty acid synthesis was 0.65 and 0.35 for lipid assembly. Double manipulation- TDCA has a value of 0.6 for fatty acid synthesis and 0.4 for lipid assembly. Taken together, these data showed that under our experimental conditions, about 60%-65% of the total metabolic flux control lay in the fatty acid synthesis group of reactions. Nevertheless because both parts of the lipid biosynthesis pathway exert significant flux control, we suggest strongly that manipulation of single enzyme will not affect the product yield appreciably.

Transformation of PHB and PHBV genes driven by maize ubiquitin promoter into oil palm for the production of biodegradable plastics

Three bacterial genes coding for the enzymes, 3-ketothiolase (bktB), acetoacetyl-CoA reductase (phaB) and PHB synthase (phaC), required for the synthesis of PHB from acetyl-CoA in bacteria were transformed into oil palm embryogenic calli. For the production of copolymer polyhydroxybutyrate–co-valerate (PHBV), the threonine dehydratase (tdcB) gene from Escherichia coli was also transformed into oil palm embryogenic calli for producing propionyl-CoA, the substrate for hydroxyvalerate. These genes were under the control of the maize ubiquitin promoter. Currently, many transformed embryogenic lines resistant to the herbicide Basta have already been produced. These transformed calli were later regenerated to produce a few hundred plantlets which are now growing in a biosafety screenhouse. Molecular analyses have demonstrated stable integration of the transgenes in their genome.

Gene discovery via expressed sequence tags from the oil palm (Elaeis guineensis Jacq.) mesocarp

Expressed sequence tags (ESTs) have been used for many applications such as to reveal gene expression patterns, gene regulation and sequence diversity. A total of 1011 unique transcripts corresponding to 1463 genes have been identified from the ESTs generated from 17-week oil palm mesocarp cDNA library. This approach was found to be successful in the discovery of new and important genes expressed in the mesocarp tissue which are associated with the various cellular processes of the tissue. It was observed that 12.9% of the total genes expressed in the 17-week mesocarp cDNA library can be categorized under metabolism. This is in agreement with the function of the tissue which is involved in many biochemical processes including amino acid and fatty acid metabolism. Most importantly are the discoveries of genes playing important roles in the fatty acid and wax biosynthesis pathway such as acetyl-CoA carboxylase, stearoyl-ACP desaturase, acyl carrier protein (ACP), lysophosphatidic acid acyltransferase, Δ6-palmitoyl-ACP desaturase and lipase. These genes can serve as targets for genetic manipulation where such endeavours have been extensively carried out in other plants such as Brassica napus and Olea europaea to help increase the economic value of the oil. Genes and protein associated with ethylene synthesis and signal transduction pathway were also identified from the 17-week mesocarp ESTs. Dot blot analysis was carried out to help in identifying potential tissuespecific genes, which can lead to the isolation of the tissue-specific promoters for manipulation of the mesocarp tissue. This is in particular to direct accumulation of transgenic products, such as new specialty oils and value-added products like pharmaceuticals and nutraceuticals to the mesocarp.

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: 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.

Carotenoid profiles and preliminary investigation on carotenoid biosynthesis in the oil palm (Elaeis guineensis) mesocarp

The changes in carotenoid content and profile were studied in oil palm (E. guineensis) mesocarp at various stages of development. Spectrophotometric analysis showed that chlorophyll synthesis predominates in the young fruits but it then shifts strongly to carotenoids as the fruit ripens. The major carotenoids in the oil from ripe fruits are α- and β-carotenes. A very wide variation in total carotenoid content was observed in various E. guineensis genotypes. High performance liquid chromatography (HPLC) analysis using both C18 and C30 stationary phases showed the C30 phase to be superior in separating the cis– and trans- isomers of both α- and β-carotenes and resolving other components. Lycopene was not detected in at all. Incorporation studies were carried out with various 14C-labelled substrates – acetate, glyceraldehyde-3-phosphate (G3P), isopentenyl, pyrophosphate (IPP), mevalonic acid (MVA) and pyruvate. IPP was the most incorporated showing it to be a major intermediate in carotenoid synthesis in the oil palm. There was also considerable incorporation of acetate, IPP and MVA into α- and β-carotenes. G3P and pyruvate were not incorporated into a- and ß-carotenes suggesting that carotenoid synthesis in the oil palm follows the acetate/ mevalonate pathway.

Activity studies, gene characterization and manipulation of β-ketothiolase of oil palm (Elaeis guineensis Jacq.) mesocarp

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.