Roohaida Othman

Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

Flavonoid biosynthesis genes putatively identified in the aromatic plant Polygonum minus via Expressed Sequences Tag (EST) analysis.

P. minus is an aromatic plant, the leaf of which is widely used as a food additive and in the perfume industry. The leaf also accumulates secondary metabolites that act as active ingredients such as flavonoid. Due to limited genomic and transcriptomic data, the biosynthetic pathway of flavonoids is currently unclear. Identification of candidate genes involved in the flavonoid biosynthetic pathway will significantly contribute to understanding the biosynthesis of active compounds. We have constructed a standard cDNA library from P. minus leaves, and two normalized full-length enriched cDNA libraries were constructed from stem and root organs in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis. Thus, large-scale sequencing of P. minus cDNA libraries identified 4196 expressed sequences tags (ESTs) which were deposited in dbEST in the National Center of Biotechnology Information (NCBI). From the three constructed cDNA libraries, 11 ESTs encoding seven genes were mapped to the flavonoid biosynthetic pathway. Finally, three flavonoid biosynthetic pathway-related ESTs chalcone synthase, CHS (JG745304), flavonol synthase, FLS (JG705819) and leucoanthocyanidin dioxygenase, LDOX (JG745247) were selected for further examination by quantitative RT-PCR (qRT-PCR) in different P. minus organs. Expression was detected in leaf, stem and root. Gene expression studies have been initiated in order to better understand the underlying physiological processes.

Identification of cDNAs for jasmonic acid-responsive genes in Polygonum minus roots by suppression subtractive hybridization

Elicitation, the plant-based biotechnology approach that utilizes the ability of plant roots to absorb and secrete a vast variety of bioactive compounds, was studied on Polygonum minus using jasmonic acid (JA) as an elicitor. To understand the overall molecular responses of P. minus roots to JA induction, a subtracted cDNA library was constructed using the suppression subtractive hybridization (SSH) method. From a total of 1,344 randomly selected colonies, 190 clones were shown to be differentially expressed using Reverse Northern hybridization. BLAST analysis revealed that clones were similar to genes associated with the biosynthesis of aromatic compounds through the oxylipin pathway, such as alcohol dehydrogenase and lipoxygenase. Putative clones involved in the shikimate pathway, including S-adenosyl-l-methionine synthetase and S-adenosyl-l-homocysteine hydrolase, were identified with predicted roles in phenylpropanoids’ biosynthesis. Genes responding to abiotic stress unique to JA elicitation, such as ELI3-1, glutathione S-transferase and peroxidase 1, were also identified. The kelch-repeat containing F-box family protein, a possible transcription factor in response to JA elicitation was also found. The results of the RT-PCR showed that the eight selected clones were strongly up-regulated, except for lipoxygenase, which showed a slightly higher expression of the transcript levels in response to the JA elicitation.

High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions

A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.

Engineering the lactococcal mevalonate pathway for increased sesquiterpene production

Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT-qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced.

Monoterpene Alcohol Metabolism: Identification, Purification, and Characterization of Two Geraniol Dehydrogenase Isoenzymes from Polygonum minus Leaves

NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).

Functional characterization of sesquiterpene synthase from polygonum minus

Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

Fruit-specific expression of papaya subtilase gene

Subtilisin-like serine proteases (EC 3.4.21) consist of a widespread family of enzymes that is involved in various processes including in plants. The full-length cDNA (CpSUB1) and the corresponding genomic DNA for papaya subtilase have been obtained using rapid amplification of cDNA ends (RACEs) and PCR primer walking techniques, respectively. The cDNA clone contains an open reading frame of 2316bp encoding 772 amino acids with a calculated molecular mass of 82.6kDa and an isoelectric point (pI) of 8.97. The CpSUB1 gene is composed of nine exons and eight introns. The amino acid sequence encoded by CpSUB1 shared high identity (>60%) with the amino acid sequence of other plant subtilisin-like proteases. Sequence analysis of CpSUB1 revealed the presence of a possible signal peptide (25 amino acid residues) and an NH(2)-terminal prosequence (88 amino acid residues). In addition, papaya subtilase possesses the characteristic subtilisin catalytic triad amino acids namely Asp, His and Ser, together with the substrate-binding site, Asn. DNA hybridization analysis showed that subtilase gene exists as a single copy in the papaya genome. RNA hybridization analyses showed that expression of the subtilase transcripts was only detected in mesocarp but not in non-fruit tissues. Gene expression in fruit tissues reached the highest level during the ripening stage at which the fruits undergo dramatic softening process. Subsequently, pro-subtilase ( approximately 80kDa) was expressed as recombinant pro-enzyme ( approximately 97kDa), which was used to generate antiserum against papaya subtilase, anti-sub. Protein gel blot analysis using anti-sub towards total protein extracted from all ripening stages revealed that a protein with a molecular mass of approximately 70kDa reacted with the antiserum. Hence both RNA hybridization and protein gel blot analyses confirmed the presence of subtilase during papaya fruit ripening, pointing to its possible involvement in this important process.

Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase

Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.

EuDBase: An online resource for automated EST analysis pipeline (ESTFrontier) and database for red seaweed Eucheuma denticulatum.

Functional genomics has proven to be an efficient tool in identifying genes involved in various biological functions. However the availability of commercially important seaweed Eucheuma denticulatum functional resources is still limited. EuDBase is the first seaweed online repository that provides integrated access to ESTs of Eucheuma denticulatum generated from samples collected from Kudat and Semporna in Sabah, Malaysia. The database stored 10,031 ESTs that are clustered and assembled into 2,275 unique transcripts (UT) and 955 singletons. Raw data were automatically processed using ESTFrontier, an in-house automated EST analysis pipeline. Data was collected in MySQL database. Web interface is implemented using PHP and it allows browsing and querying EuDBase through search engine. Data is searchable via BLAST hit, domain search, Gene Ontology or KEGG Pathway. A user-friendly interface allows the identification of sequences either using a simple text query or similarity search. The development of EuDBase is initiated to store, manage and analyze the E. denticulatum ESTs and to provide accumulative digital resources for the use of global scientific community. EuDBase is freely available from http://www.inbiosis.ukm.my/eudbase/.