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Fulltext Enhancing flavonoid production by promiscuous activity of prenyltransferase, BrPT2 from Boesenbergia rotunda

Liew YJM, Lee YK, Khalid N, Rahman NA, Tan BC

PeerJ, 2020;8:e9094.
PMID: 32391211 DOI: 10.7717/peerj.9094

Flavonoids and prenylated flavonoids are active components in medicinal plant extracts which exhibit beneficial effects on human health. Prenylated flavonoids consist of a flavonoid core with a prenyl group attached to it. This prenylation process is catalyzed by prenyltranferases (PTs). At present, only a few flavonoid-related PT genes have been identified. In this study, we aimed to investigate the roles of PT in flavonoid production. We isolated a putative PT gene (designated as BrPT2) from a medicinal ginger, Boesenbergia rotunda. The deduced protein sequence shared highest gene sequence homology (81%) with the predicted homogentisate phytyltransferase 2 chloroplastic isoform X1 from Musa acuminata subsp. Malaccensis. We then cloned the BrPT2 into pRI vector and expressed in B. rotunda cell suspension cultures via Agrobacterium-mediated transformation. The BrPT2-expressing cells were fed with substrate, pinostrobin chalcone, and their products were analyzed by liquid chromatography mass spectrometry. We found that the amount of flavonoids, namely alpinetin, pinostrobin, naringenin and pinocembrin, in BrPT2-expressing cells was higher than those obtained from the wild type cells. However, we were unable to detect any targeted prenylated flavonoids. Further in-vitro assay revealed that the reaction containing the BrPT2 protein produced the highest accumulation of pinostrobin from the substrate pinostrobin chalcone compared to the reaction without BrPT2 protein, suggesting that BrPT2 was able to accelerate the enzymatic reaction. The finding of this study implied that the isolated BrPT2 may not be involved in the prenylation of pinostrobin chalcone but resulted in high yield and production of other flavonoids, which is likely related to enzyme promiscuous activities.
Cell-Free Expression of a Plant Membrane Protein BrPT2 From Boesenbergia Rotunda

Liew YJM, Lee YK, Khalid N, Rahman NA, Tan BC

Mol Biotechnol, 2021 Apr;63(4):316-326.
PMID: 33565047 DOI: 10.1007/s12033-021-00304-z

Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.
Fulltext Complete chloroplast genome of Boesenbergia rotunda and a comparative analysis with members of the family Zingiberaceae

Liew YJM, Chua KO, Yong HS, Song SL, Chan KG

Rev Bras Bot, 2022;45(4):1209-1222.
PMID: 36320930 DOI: 10.1007/s40415-022-00845-w

Boesenbergia rotunda (L.) Mansf. is a medically important ginger species of the family Zingiberaceae but its genomic information on molecular phylogeny and identification is scarce. In this work, the chloroplast genome of B. rotunda was sequenced, characterized and compared to the other Zingiberaceae species to provide chloroplast genetic resources and to determine its phylogenetic position in the family. The chloroplast genome of B. rotunda was 163,817 bp in length and consisted of a large single-copy (LSC) region of 88,302 bp, a small single-copy (SSC) region of 16,023 bp and a pair of inverted repeats (IRA and IRB) of 29,746 bp each. The chloroplast genome contained 113 unique genes, including 79 protein-coding genes, 30 transfer RNA (tRNA) genes and four ribosomal RNA (rRNA) genes. Several genes had atypical start codons, while most amino acids exhibited biased usage of synonymous codons. Comparative analyses with various chloroplast genomes of Zingiberaceae taxa revealed several highly variable regions (psbK-psbI, trnT-GGU-psbD, rbcL-accD, ndhF-rpl32, and ycf1) in the LSC and SSC regions in the chloroplast genome of B. rotunda that could be utilized as molecular markers for DNA barcoding and species delimitation. Phylogenetic analyses based on shared protein-coding genes revealed that B. rotunda formed a distinct lineage with B. kingii Mood & L.M.Prince, in a subclade that also contained the genera Kaempferia and Zingiber. These findings constitute the first chloroplast genome information of B. rotunda that could be a reference for phylogenetic analysis and identification of genus Boesenbergia within the Zingiberaceae family.
SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40415-022-00845-w.
Unravelling the Significance of Seed Proteomics: Insights into Seed Development, Function, and Agricultural Applications

Al-Obaidi JR, Lau SE, Liew YJM, Tan BC, Rahmad N

Protein J, 2024 Dec;43(6):1083-1103.
PMID: 39487361 DOI: 10.1007/s10930-024-10240-x

Seeds are essential for plant reproduction, ensuring species survival and dispersal while adapting to diverse environments throughout a plant's life. Proteomics has emerged as a powerful tool for deciphering the complexities of seed growth, germination, and stress responses. Advanced proteomic technologies enable the analysis of protein changes during germination, dormancy, and ageing, enhancing our understanding of seed lifespan and vitality. Recent studies have revealed detailed insights into metabolic processes and storage protein profiles across various plant species. This knowledge is crucial for improving seed storage, conserving quality, and maintaining viability. Additionally, it contributes to sustainable agriculture by identifying stress-responsive proteins and signalling pathways that can mitigate stress and enhance farming practices. This review highlights significant advancements in seed proteomics over the past decade, discussing critical discoveries related to storage proteins, protein interactions, and proteome modifications due to stress. It illustrates how these insights transform seed biology, boosting productivity, food security, and environmentally friendly practices. The review also identifies existing knowledge gaps and provides direction for future research, underscoring the need for continued interdisciplinary collaboration in this dynamic field.
Whole genome analysis revealed the role of blaOXA-23 and blaOXA-66 genes in carbapenem resistance of Acinetobacter baumannii strains

Mat Ghani N, Hong KW, Liew YJM, Lau YY, Yong HS, Tee KK, et al.

Pathog Glob Health, 2025;119(1-2):10-21.
PMID: 39699991 DOI: 10.1080/20477724.2024.2442194

Acinetobacter baumannii is a multidrug-resistant bacterium that has emerged as a significant nosocomial pathogen globally and renowned for its ability to acquire antimicrobial resistance (AMR) genes. However, understanding of its resistance mechanisms to certain drug classes remains limited. This study focused on four bacterial strains (AB863, AB889, AB930, and AB960) exhibiting carbapenem resistance. They demonstrated high minimum inhibitory concentration (MIC) (128 mg/L) to meropenem and were categorized as extensively drug-resistant strains. Subsequently, they were identified as A. baumannii through 16S rRNA gene sequence analysis and species-specific PCR targeting the blaOXA51-like gene. Three strains were sequenced for their genomes to study the genetic determinants and functional relevance of carbapenem resistance. The draft genome length of the strains ranged from 3.8 to 4.0 Mbp. A total of 16 antibiotic resistance genes including the genes blaOXA-23 and blaOXA-66 which mediate carbapenem resistance were identified in the genomes. A comprehensive multilocus sequence typing analysis involving 95 A. baumannii strains from different Asian countries assigned the four strains to sequence type 2 (ST2), the most predominant ST circulating in Asia. Comparative genome analysis also revealed blaOXA-66 as the most dominant variant of blaOXA-51-like gene and also a widespread distribution of blaOXA-23 gene. In addition, various mobile genetic elements associated with AMR genes and three efflux pumps families were detected in the genomes of the strains. Transformation of blaOXA-23 and blaOXA-66 genes resulted in meropenem resistance in the transformant which exhibited a MIC of 2 mg/L, thus confirming direct involvement of both genes in carbapenem resistance.