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Characterization of a KCS-like KASII from Jessenia bataua that elongates saturated and monounsaturated stearic acids in Arabidopsis thaliana

Teh OK, Ramli US

Mol Biotechnol, 2011 Jun;48(2):97-108.
PMID: 21113689 DOI: 10.1007/s12033-010-9350-x

As the world population grows, the demand for food increases. Although vegetable oils provide an affordable and rich source of energy, the supply of vegetable oils available for human consumption is limited by the "fuel vs food" debate. To increase the nutritional value of vegetable oil, metabolic engineering may be used to produce oil crops of desirable fatty acid composition. We have isolated and characterized β-ketoacyl ACP-synthase II (KASII) cDNA from a high-oleic acid palm, Jessenia bataua. Jessenia KASII (JbKASII) encodes a 488-amino acid polypeptide that possesses conserved domains that are necessary for condensing activities. When overexpressed in E. coli, recombinant His-tagged JbKASII was insoluble and non-functional. However, Arabidopsis plants expressing GFP-JbKASII fusions had elevated levels of arachidic acid (C20:0) and erucic acid (C22:1) at the expense of stearic acid (C18:0) and oleic acid (C18:1). Furthermore, JbKASII failed to complement the Arabidopsis KASII mutant, fab1-2. This suggests that the substrate specificity of JbKASII is similar to that of ketoacyl-CoA synthase (KCS), which preferentially elongates stearic and oleic acids, and not palmitic acid. Our results suggest that the KCS-like JbKASII may elongate C18:0 and C18:1 to yield C20:0 and C22:1, respectively. JbKASII may, therefore, be an interesting candidate gene for promoting the production of very long chain fatty acids in transgenic oil crops.
Application of Proteomics Technologies in Oil Palm Research

Lau BYC, Othman A, Ramli US

Protein J, 2018 12;37(6):473-499.
PMID: 30367348 DOI: 10.1007/s10930-018-9802-x

Proteomics technologies were first applied in the oil palm research back in 2008. Since proteins are the gene products that are directly correspond to phenotypic traits, proteomic tools hold a strong advantage above other molecular tools to comprehend the biological and molecular mechanisms in the oil palm system. These emerging technologies have been used as non-overlapping tools to link genome-wide transcriptomics and metabolomics-based studies to enhance the oil palm yield and quality through sustainable plant breeding. Many efforts have also been made using the proteomics technologies to address the oil palm's Ganoderma disease; the cause and management. At present, the high-throughput screening technologies are being applied to identify potential biomarkers involved in metabolism and cellular development through determination of protein expression changes that correlate with oil production and disease. This review highlights key elements in proteomics pipeline, challenges and some examples of their implementations in plant studies in the context of oil palm in particular. We foresee that the proteomics technologies will play more significant role to address diverse issues related to the oil palm in the effort to improve the oil crop.
Use of control analysis to study the regulation of plant lipid biosynthesis

Ramli US, Baker DS, Quant PA, Harwood JL

Biochem Soc Trans, 2002 Nov;30(Pt 6):1043-6.
PMID: 12440968

Control analysis is a powerful method to quantify the regulation of metabolic pathways. We have applied it to lipid biosynthesis for the first time by using model tissue culture systems from the important oil crops, olive ( Olea europaea L.) and oil palm ( Elaeis guineensis Jacq.). By the use of top-down control analysis, fatty acid biosynthesis has been shown to exert more control than lipid assembly under different experimental conditions. However, both parts of the lipid biosynthetic pathway are important, so that attempts to alter oil yield by manipulating the activity of a single enzyme step are very unlikely to produce significant increases.
Deciphering key proteins of oil palm (Elaeis guineensis Jacq.) fruit mesocarp development by proteomics and chemometrics

Hassan H, Amiruddin MD, Weckwerth W, Ramli US

Electrophoresis, 2019 01;40(2):254-265.
PMID: 30370930 DOI: 10.1002/elps.201800232

Palm oil is an edible vegetable oil derived from lipid-rich fleshy mesocarp tissue of oil palm (Elaeis guineensis Jacq.) fruit and is of global economic and nutritional relevance. While the understanding of oil biosynthesis in plants is improving, the fundamentals of oil biosynthesis in oil palm still require further investigations. To gain insight into the systemic mechanisms that govern oil synthesis during oil palm fruit ripening, the proteomics approach combining gel-based electrophoresis and mass spectrometry was used to profile protein changes and classify the patterns of protein accumulation during these complex physiological processes. Protein profiles from different stages of fruit ripening at 10, 12, 14, 15, 16, 18 and 20 weeks after anthesis (WAA) were analysed by two-dimensional gel electrophoresis (2DE). The proteome data were then visualised using a multivariate statistical analysis of principal component analysis (PCA) to get an overview of the proteome changes during the development of oil palm mesocarp. A total of 68 differentially expressed protein spots were successfully identified by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF/TOF) and functionally classified using ontology analysis. Proteins related to lipid production, energy, secondary metabolites and amino acid metabolism are the most significantly changed proteins during fruit development representing potential candidates for oil yield improvement endeavors. Data are available via ProteomeXchange with identifier PXD009579. This study provides important proteome information for protein regulation during oil palm fruit ripening and oil synthesis.
Fulltext Method developments to extract proteins from oil palm chromoplast for proteomic analysis

Lau BY, Deb-Choudhury S, Morton JD, Clerens S, Dyer JM, Ramli US

Springerplus, 2015;4:791.
PMID: 26702380 DOI: 10.1186/s40064-015-1576-4

Proteins from the plant chromoplast are essential for many physiological processes such as fatty acid biosynthesis. Different protein extraction methods were tested to find the most robust method to obtain oil palm chromoplast proteins for mass spectrometry analysis. Initially, two different solvents were employed to reduce the fruit lipids. Then, two plant cell wall digestive enzymes were used to acquire the protoplasts to increase the protein extraction effectiveness. A two-stage centrifugation-based fractionation approach enhanced the number of identified proteins, particularly the fatty acid biosynthetic enzymes. The effectiveness of each extraction method was assessed using protein yields and 2DE gel profiles. The ideal method was successfully used to establish the 2DE chromoplast proteome maps of low and high oleic acid mesocarps of oil palm. Further nanoLC-MS/MS analysis of the extracted chromoplast proteins led to the identification of 162 proteins, including some of the main enzymes involved in the fatty acid biosynthesis. The established procedures would provide a solid foundation for further functional studies, including fatty acid biosynthetic expression profiling and evaluation of regulatory function.
Characterization of apigenin and luteolin derivatives from oil palm (Elaeis guineensis Jacq.) leaf using LC-ESI-MS/MS

Tahir NI, Shaari K, Abas F, Parveez GK, Ishak Z, Ramli US

J Agric Food Chem, 2012 Nov 14;60(45):11201-10.
PMID: 23116142 DOI: 10.1021/jf303267e

The palm oil industry generates several byproducts, and more than half of the dry weight of the waste is of oil palm leaf whereby the tissue is underutilized. Recently, several research studies found promising potential of oil palm fronds as a source of nutraceutical due to its bioactive properties. However, the chemical composition of the tissue is still not deciphered. Using reversed-phase liquid chromatography (LC) electrospray mass spectrometry (ESI-MS), glycosylated apigenin and luteolin were separated and identified from oil palm (Elaeis guineensis Jacq.) leaf and structures of the constituents were elucidated by collision-induced dissociation (CID) tandem MS. From 28 derivatives of the flavones, 9 compounds were conjugated with hydroxymethylglutaric (HMG) acid. Improved knowledge on oil palm especially on bioactive component of the leaf tissue will allow correlation of its beneficial effects and further promotes efficient utilization of this agriculture byproduct.
Application of a Mass Spectrometric Approach to Detect the Presence of Fatty Acid Biosynthetic Phosphopeptides

Lau BY, Clerens S, Morton JD, Dyer JM, Deb-Choudhury S, Ramli US

Protein J, 2016 Apr;35(2):163-70.
PMID: 26993480 DOI: 10.1007/s10930-016-9655-0

The details of plant lipid metabolism are relatively well known but the regulation of fatty acid production at the protein level is still not understood. Hence this study explores the importance of phosphorylation as a mechanism to control the activity of fatty acid biosynthetic enzymes using low and high oleic acid mesocarps of oil palm fruit (Elaeis guineensis variety of Tenera). Adaptation of neutral loss-triggered tandem mass spectrometry and selected reaction monitoring to detect the neutral loss of phosphoric acid successfully found several phosphoamino acid-containing peptides. These peptides corresponded to the peptides from acetyl-CoA carboxylase and 3-enoyl-acyl carrier protein reductase as identified by their precursor ion masses. These findings suggest that these enzymes were phosphorylated at 20th week after anthesis. Phosphorylation could have reduce their activities towards the end of fatty acid biosynthesis at ripening stage. Implication of phosphorylation in the regulation of fatty acid biosynthesis at protein level has never been reported.
Comparative analysis of statistical tools for oil palm phytochemical research

Ishak NA, Tahir NI, Mohd Sa'id SN, Gopal K, Othman A, Ramli US

Heliyon, 2021 Feb;7(2):e06048.
PMID: 33553773 DOI: 10.1016/j.heliyon.2021.e06048

Recent advances in phytochemical analysis have allowed the accumulation of data for crop researchers due to its capacity to footprint and distinguish metabolites that are present within an organisms, tissues or cells. Apart from genotypic traits, slight changes either by biotic or abiotic stimuli will have significant impact on the metabolite abundances and will eventually be observed through physicochemical characteristics. Apposite data mining to interpret the mounds of phytochemical information from such a dynamic system is thus incumbent. In this investigation, several statistical software platforms ranging from exploratory and confirmatory technique of multivariate data analysis from four different statistical tools of COVAIN, SIMCA-P+, MetaboAnalyst and RIKEN Excel Macro were appraised using an oil palm phytochemical data set. As different software tool encompasses its own advantages and limitations, the insights gained from this assessment were documented to enlighten several aspects of functions and suitability for the adaptation of the tools into the oil palm phytochemistry pipeline. This comparative analysis will certainly provide scientists with salient notes on data assessment and data mining that will later allow the depiction of the overall oil palm status in-situ and ex-situ.
Fulltext Sustainable Palm Oil-The Role of Screening and Advanced Analytical Techniques for Geographical Traceability and Authenticity Verification

Ramli US, Tahir NI, Rozali NL, Othman A, Muhammad NH, Muhammad SA, et al.

Molecules, 2020 Jun 25;25(12).
PMID: 32630515 DOI: 10.3390/molecules25122927

Palm oil production from oil palm (Elaeis guineensis Jacq.) is vital for the economy of Malaysia. As of late, sustainable production of palm oil has been a key focus due to demand by consumer groups, and important progress has been made in establishing standards that promote good agricultural practices that minimize impact on the environment. In line with the industrial goal to build a traceable supply chain, several measures have been implemented to ensure that traceability can be monitored. Although the palm oil supply chain can be highly complex, and achieving full traceability is not an easy task, the industry has to be proactive in developing improved systems that support the existing methods, which rely on recorded information in the supply chain. The Malaysian Palm Oil Board (MPOB) as the custodian of the palm oil industry in Malaysia has taken the initiative to assess and develop technologies that can ensure authenticity and traceability of palm oil in the major supply chains from the point of harvesting all the way to key downstream applications. This review describes the underlying framework related to palm oil geographical traceability using various state-of-the-art analytical techniques, which are also being explored to address adulteration in the global palm oil supply chain.
Fulltext Use of headspace-gas chromatography-ion mobility spectrometry to detect volatile fingerprints of palm fibre oil and sludge palm oil in samples of crude palm oil

Othman A, Goggin KA, Tahir NI, Brodrick E, Singh R, Sambanthamurthi R, et al.

BMC Res Notes, 2019 Apr 16;12(1):229.
PMID: 30992056 DOI: 10.1186/s13104-019-4263-7

OBJECTIVE: The addition of residual oils such as palm fibre oil (PFO) and sludge palm oil (SPO) to crude palm oil (CPO) can be problematic within supply chains. PFO is thought to aggravate the accumulation of monochloropropanediols (MCPDs) in CPO, whilst SPO is an acidic by-product of CPO milling and is not fit for human consumption. Traditional targeted techniques to detect such additives are costly, time-consuming and require highly trained operators. Therefore, we seek to assess the use of gas chromatography-ion mobility spectrometry (GC-IMS) for rapid, cost-effective screening of CPO for the presence of characteristic PFO and SPO volatile organic compound (VOC) fingerprints.
RESULTS: Lab-pressed CPO and commercial dispatch tank (DT) CPO were spiked with PFO and SPO, respectively. Both additives were detectable at concentrations of 1% and 10% (w/w) in spiked lab-pressed CPO, via seven PFO-associated VOCs and 21 SPO-associated VOCs. DT controls could not be distinguished from PFO-spiked DT CPO, suggesting these samples may have already contained low levels of PFO. DT controls were free of SPO. SPO was detected in all SPO-spiked dispatch tank samples by all 21 of the previously distinguished VOCs and had a significant fingerprint consisting of four spectral regions.