Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Functional Characterisation of Highly Conserved and Structurally Prominent Residues of 2-Methylisoborneol Synthase.

2-Methylisoborneol is a widespread musty odourant that is produced by many bacteria including actinomycetes, cyanobacteria and myxobacteria. Two 2-methylisoborneol synthases (MIBS) that are phylogenetically distant to the known enzyme from Streptomyces coelicolor were found to be highly active for 2-methylisoborneol biosynthesis. Based on the enzyme structure and on an amino acid sequence alignment, the MIBS from S. coelicolor was extensively studied through site-directed mutagenesis.

Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli.

The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPSS488T mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPSF612A variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.

New class of hantaan virus inhibitors based on conjugation of the isoindole fragment to (+)-camphor or (-)-fenchone hydrazonesv.

This work presents the design and synthesis of camphor, fenchone, and norcamphor N-acylhydrazone derivatives as a new class of inhibitors of the Hantaan virus, which causes haemorrhagic fever with renal syndrome (HFRS). A cytopathic model was developed for testing chemotherapeutics against the Hantaan virus, strain 76-118. In addition, a study of the antiviral activity was carried out using a pseudoviral system. It was found that the hit compound possesses significant activity (IC50 = 7.6 ± 2 µM) along with low toxicity (CC50 > 1000 µM). Using molecular docking procedures, the binding with Hantavirus nucleoprotein was evaluated and the correlation between the structure of the synthesised compounds and the antiviral activity was established.

Limitation of nutrients stimulates musty odor production by Streptomyces sp. isolated from a tropical environment.

Musty odor production by actinomycetes is usually related to the presence of geosmin and 2-methylisoborneol (2-MIB), which are synthesized by enzymes encoded by the geoA and tpc genes, respectively. Streptomyces spp. strain S10, which was isolated from a water reservoir in Malaysia, has the ability to produce geosmin when cultivated in a basal salt (BS) solid medium, but no 2-MIB production occurred during growth in BS medium. Strain S10 could produce higher levels of geosmin when the phosphate concentration was limited to 0.05 mg/L, with a yield of 17.53 ± 3.12 ✕ 105 ng/L, compared with growth in BS medium. Interestingly, 2-MIB production was suddenly detected when the nitrate concentration was limited to 1.0 mg/L, with a yield of 1.4 ± 0.11 ✕ 105 ng/L. Therefore, it was concluded that phosphate- and nitrate-limiting conditions could induce the initial production of geosmin and 2-MIB by strain S10. Furthermore, a positive amplicon of geoA was detected in strain S10, but no tpc amplicon was detected by PCR analysis. Draft genome sequence analysis showed that one open reading frame (ORF) contained a conserved motif of geosmin synthase with 95% identity with geoA in Streptomyces coelicolor A3 (2). In the case of the tpc genes, it was found that one ORF showed 23% identity to the known tpc gene in S. coelicolor A3(2), but strain S10 lacked one motif in the N-terminus.

Borneol protects against cerulein-induced oxidative stress and inflammation in acute pancreatitis mice model.

Borneol is a commonly used flavouring substance in traditional Chinese medicine, which possesses several pharmacological activities including analgesic, antiinflammatory, and antioxidant properties. The aim of this study was to investigate the effects of borneol on cerulein-induced acute pancreatitis (AP) model. Swiss albino mice were pretreated with borneol (100 and 300 mg/kg) daily for 7 days, before six consecutive injections of cerulein (50 µg/kg/hr, intraperitoneally). The protective effect of borneol was studied by biochemical, enzyme linked immunosorbent assay, histological, immunoblotting, and immunohistochemical analysis. Oral administration of borneol significantly attenuated pancreatic damage by reducing amylase, lipase levels and histological changes. Borneol attenuated cerulein-induced oxidative-nitrosative stress by decreasing malondialdehyde, nitrite levels, and elevating reduced glutathione levels. Pancreatic inflammation was ameliorated by inhibiting myeloperoxidase activity and pro-inflammatory cytokine (Interleukins and TNF-α) levels. Furthermore, borneol administration significantly increased nuclear factor E2-related factor 2 (Nrf2), superoxide dismutase (SOD1) expression and reduced phospho-NF-κB p65 expression. Treatment with borneol significantly inhibited TNF-α, IL-1ß, IL-6, and inducible nitric oxide synthase expression in cerulein-induced AP mouse model. Together, these results indicate that borneol which is currently used as US-FDA approved food adjuvant has the potential to attenuate cerulein-induced AP possibly by reducing the oxidative damage and pancreatic inflammation by modulating Nrf2/NF-κB pathway.

Production of optically pure (-)-borneol by Pseudomonas monteilii TCU-CK1 and characterization of borneol dehydrogenase involved.

(-)-Borneol is a bicyclic plant secondary metabolite. Optically pure (-)-borneol can only be obtained from plants, and demand exceeds supply in China. In contrast, chemically synthesized borneol contains four different stereoisomers. A strain of Pseudomonas monteilii TCU-CK1, isolated in Hualien, Taiwan, can accumulate (-)-borneol in growth culture and selectively degrades the other three isomers when chemically synthesized borneol is used as sole carbon source. This (-)-borneol production method can be scaled-up for production of large quantities in the future. More importantly, laborious plant cultivation and harvest is no longer required. The main enzyme that appears in this degradation pathway, borneol dehydrogenase (BDH), and the genome sequence of TCU-CK1 are reported. The kcat/Km values of TCU-CK1 BDH on (+)- and (-)-borneol are 538.4 ± 38.4 and 17.7 ± 1.1 (s-1 mM-1), respectively. About ∼30 fold difference in the kcat/Km value between (+)-borneol and (-)-borneol was observed, in good agreement with the fact that TCU-CK1 prefers to degrade (+)-borneol, rather than (-)-borneol. A BDH isozyme was identified in a strain in which the primary BDH gene had been knocked out. (-)-Camphor can work as an inhibitor of BDH with a Ki of 1.03 ± 0.11 mM at pH 7.0, leading to the accumulation of (-)-borneol in culture. (Patent pending).

Effects of exogenous application of citrulline on prolonged water stress damages in hyssop (Hyssopus officinalis L.): Antioxidant activity, biochemical indices, and essential oils profile.

This study examined the effects of exogenous citrulline (control, 1 and 2 mM) and water availability (100%, 50% and 25% WA) on antioxidant attributes and essential oil constituents of Hyssopus officinalis L. in two successive harvests. Hyssop tolerantly responded to water deficiency by well-promoted antioxidant enzymes (i.e., superoxide dismutase, and catalase), strong DPPH-scavenging activity, and increasing polyphenols; however, the essential oil content was negatively reduced by water stress. External citrulline further increased the activity of antioxidant enzymes. Citrulline application at 2 mM under severe water stress could also improve essential oil (EO) content in the first and second harvests by about 15 and 30%, respectively. Furthermore, under severe drought, citrulline at 2 mM could obtain the highest yield of isopinocamphone (47%) as the main component of EO. The results showed the high potential of this novel applied metabolite agent to be used in a well-fulfilled production of this medicinal plant.

Multiscale study on the enhancing effect and mechanism of borneolum on transdermal permeation of drugs with different log P values and molecular sizes.

D-borneolum is commonly used as a permeation enhancer in Traditional Chinese Medicine (TCM) formulas for transdermal application. Additionally, two other sources of borneolums were recorded in the 2015 edition of the Chinese Pharmacopoeia (ChP), including L-borneolum and borneolum syntheticum. To guide the selection and application of borneolum, the safety and enhancing effect of three sources of borneolums were investigated on transdermal permeation of compounds with different octanol-water partition coefficient (log P) values and molecular weights (MWs). Both the results of cellular cytotoxicity and in vitro transdermal permeation experiments showed that all three sources of borneolums could be applied in TDDS as permeation enhancers. Moreover, all three sources of borneolums achieved optimal permeation-enhancing performances on transdermal drugs with lower log P values as well as higher MWs. Further study was carried out to elucidate the potential molecular mechanism of borneolum enhancing transdermal drug delivery via transmission electron microscope (TEM) and coarse-grained molecular dynamic (CG-MD) simulation. Borneolum significantly promoted transdermal delivery of drugs via changing the dense morphology of the stratum corneum (SC), disturbing the ordered arrangement of ceramide (CER) and free fatty acid (FFA) molecules in lipid layers, and further increasing the diffusion rate of drugs in the lipid layers.

Effect of different doses of borneol on the pharmacokinetics of vinpocetine in rat plasma and brain after intraocular administration.

The effect of different doses of borneol on the pharmacokinetics of vinpocetine after intraocular administration in the rat plasma and the brain was investigated.Intraocular administration of vinpocetine (3 mg/kg) was performed, in combination with different doses (0, 5, 10, and 20 mg/kg) of borneol. Intravenous administration of vinpocetine was used as a control (1 mg/kg). The concentrations of vinpocetine in the rat plasma and the brain were determined using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Using the non-compartmental models with the DSA 2.0 software, the main pharmacokinetics parameters and the brain-targeting effect evaluated.In comparison with intravenous administration, after intraocular administration of vinpocetine alone, the absolute bioavailability (F) of vinpocetine was 43.82% for the plasma, and the drug target index (DTI) was 1.05 for the brain. After intraocular administration of vinpocetine combined with different doses of borneol, the relative bioavailability (Fr) of vinpocetine in the plasma was increased by 130.46-182.90%. The relative bioavailability (Fr) of vinpocetine in the brain was improved (147.19-225.36%). The DTI was 1.12, 1.18, and 1.21 for 5, 10, and 20 mg/kg of borneol, respectively.Compared with the intraocular administration of vinpocetine alone, the co-administration of different doses of borneol resulted in an obvious brain targeting effect.

A fluorescence-displacement assay using molecularly imprinted polymers for the visual, rapid, and sensitive detection of the algal metabolites, geosmin and 2-methylisoborneol.

A visual, rapid, and sensitive method for the detection of two algal metabolites, geosmin (GSM) and 2-methylisoborneol (2-MIB) using a competitive displacement technique based on molecular imprinted polymers (MIPs) and fluorescent tags was developed. In this method, fluorescent tags that bind to synthetic receptor sites of MIPs were designed and synthesised. In the presence of target analytes (geosmin and 2-methylisoborneol respectively), the tags are displaced leading to fluorescence signals. The MIPs were derived from the polymerisation of functional monomers and crosslinkers in the presence of suitable templates. Good to high binding capacities and selectivities were obtained with the MIPs. The displacement of fluorescent-tagged substrates from the respective MIPs by the target analytes enabled the quantitative detection of geosmin at concentrations as low as 0.38 µM (69 µg L-1), while the LOD for 2-methylisoborneol is 0.29 µM (48 µg L-1) without any cross-reactivity, non-specific (false-positive) binding, and matrix complications. Qualitative detection of geosmin and 2-methylisoborneol is also possible via visualisation of fluorescence using a hand held UV lamp, with LOD for geosmin and 2-methylisoborneol at 0.44 µM (80 µg L-1) and 0.35 µM (60 µg L-1), respectively. The sensitivity of the system can be improved with a pre-concentration step using the respective MIPs as a sorbent.

Design, characterization and comparison of transdermal delivery of colchicine via borneol-chemically-modified and borneol-physically-modified ethosome.

Gout is a kind of joint disease characterized by the accumulation of monosodium urate (MSU) crystals in the joint and its surrounding tissue, causing persistent hyperuricemia. Colchicine is the first choice of treatment for acute gout attacks. Due to strong toxicity of colchicines oral tablets, there are high fluctuations of blood drug concentration and serious irritation of gastrointestinal tract, and hence a transdermal preparation can help to slow down the blood drug concentration, reduce the frequency of drug taking, and improve the patients' compliance of the drug. The ethosome is a lipid carrier with high concentration of ethanol and has been proved to promote the penetration of drugs into the skin. Borneol (BO) is an excellent penetration enhancer in Chinese medicine, which can promote the entry of drugs into the skin. This paper prepared the borneol-physically-modified colchicine ethosome (COL-bpES) and used the prepared borneol-dioleoyl phosphoethanloamine (BO-DOPE) to prepare borneol-chemically-modified colchicine ethosome (COL-bcES). Compared to the free colchicine aqueous solution (free COL) and normal colchicine ethosome (COL-ES), the borneol-modified colchicine ethosome (COL-bES) demonstrated better drug penetration effect, while the particle size of the COL-bcES was lower than that of the COL-bpES. Toxicity, in vitro diffusion, pharmacokinetics and pharmacodynamics are superior to those of COL-bpES, providing a better delivery system for the treatment of small molecule inflammatory drugs.

Metabolite identification of tanshinol borneol ester in rats by high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry.

Tanshinol borneol ester (DBZ) is a potential drug candidate composed of danshensu and borneol. It shows anti-ischemic and anti-atherosclerosis activity. However, little is known about its metabolism in vivo. This research aimed to elucidate the metabolic profile of DBZ through analyzing its metabolites using high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry. Chromatographic separation was performed on an Agilent TC-C18 column (150 × 4.6 mm, 5.0 µm) with gradient elution using methanol and water containing 0.2% (v/v) formic acid as the mobile phase. Metabolite identification involved analyzing the retention behaviors, changes in molecular weights and MS/MS fragment patterns of DBZ and its metabolites. As a result, 20 potential metabolites were detected and tentatively identified in rat plasma, urine and feces after administration of DBZ. DBZ could be metabolized to O-methylated DBZ, DBZ-O-glucuronide, O-methylated DBZ-O-glucuronide, hydroxylated DBZ and danshensu. Danshensu, a hydrolysis product of DBZ, could further be transformed into 12 metabolites. The proposed method was confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of DBZ and providing valuable information on its druggability.

Permeation-enhancing effects and mechanisms of borneol and menthol on ligustrazine: A multiscale study using in vitro and coarse-grained molecular dynamics simulation methods.

Borneol (BO) and menthol (MEN) are two widely used natural permeation enhancers in the transdermal drug delivery system. In previous studies, their permeation enhancement effects and mechanisms of action on the hydrophobic drug osthole (logP = 3.8) and hydrophilic drug 5-fluorouracil (logP = -0.9) have been studied. In this study, ligustrazine (LTZ), whose logP is 1.3, was used as a model drug to provide a comprehensive understanding of the influence of its logP on the permeation-enhancing effects of BO and MEN. Both BO and MEN enhanced the permeation of LTZ through the skin stratum corneum, as determined using the modified Franz diffusion cell experiment. The enhancement mechanisms were illustrated by coarse-grained molecular dynamics simulations as follows: at low concentrations, the enhancing ratio of MEN was higher than that of BO because of the stronger perturbation effects of MEN on the lipid bilayer, making it looser and facilitating LTZ diffusion. However, at high concentrations, in addition to the diffusion mechanism, BO induced the formation of water channels to improve the permeation of LTZ; however, MEN had no significant effects through this mechanism. Their results were different from those found with osthole and 5-fluorouracil and have been discussed in this study.

Heterologous expression of 2-methylisoborneol / 2 methylenebornane biosynthesis genes in Escherichia coli yields novel C11-terpenes.

The structural diversity of terpenoids is limited by the isoprene rule which states that all primary terpene synthase products derive from methyl-branched building blocks with five carbon atoms. With this study we discover a broad spectrum of novel terpenoids with eleven carbon atoms as byproducts of bacterial 2-methylisoborneol or 2-methylenebornane synthases. Both enzymes use 2-methyl-GPP as substrate, which is synthesized from GPP by the action of a methyltransferase. We used E. coli strains that heterologously produce different C11-terpene synthases together with the GPP methyltransferase and the mevalonate pathway enzymes. With this de novo approach, 35 different C11-terpenes could be produced. In addition to eleven known compounds, it was possible to detect 24 novel C11-terpenes which have not yet been described as terpene synthase products. Four of them, 3,4-dimethylcumene, 2-methylborneol and the two diastereomers of 2-methylcitronellol could be identified. Furthermore, we showed that an E. coli strain expressing the GPP-methyltransferase can produce the C16-terpene 6-methylfarnesol which indicates the condensation of 2-methyl-GPP and IPP to 6-methyl-FPP by the E. coli FPP-synthase. Our study demonstrates the broad range of unusual terpenes accessible by expression of GPP-methyltransferases and C11-terpene synthases in E. coli and provides an extended mechanism for C11-terpene synthases.

Chemical composition and α-amylase inhibitory activity of the essential oil from Sabina chinensis cv. Kaizuca leaves.

Sabina chinensis cv. Kaizuca (SCK) is a variant of S. chinensis L. The essential oil from its leaves exhibited α-amylase inhibitory activity in vitro and the IC50 value was 187.08 ± 0.56 µg/mL. Nineteen compounds were identified from this essential oil by gas chromatography-mass spectrometry (GC-MS) analysis. The major compounds identified were bornyl acetate (42.6%), elemol (20.5%), ß-myrcene (13.7%) and ß-linalool (4.0%). In order to study the reason of the α-amylase inhibitory activity of this essential oil, the identified compounds were docked with α-amylase by molecular docking individually. Among these compounds, γ-eudesmol exhibited the lowest binding energy (-6.73 kcal/mol), followed by α-copaen-11-ol (-6.66 kcal/mol), cubedol (-6.39 kcal/mol) and α-acorenol (-6.12 kcal/mol). The results indicated that these compounds were the active ingredients responsible for the α-amylase inhibitory activity of essential oil from SCK.

Characterization of Musty Odor-Producing Actinomycetes from Tropics and Effects of Temperature on the Production of Musty Odor Compounds.

Geosmin and 2-methylisoborneol (MIB) outbreaks in tropical water bodies, such as Southeast Asia, by actinomycetes have not yet been elucidated in detail. Six Streptomyces isolates from lowland environments in Malaysia were selected and evaluated for their odor production under different temperatures. The gene responsible for the production of geosmin, geoA, was detected in all isolates, while only two isolates harbored tpc, which is responsible for 2-MIB production. This result suggested that geosmin and 2-MIB synthesis pathway genes already existed in the environment in the Tropics of Southeast Asia. Furthermore, our isolates produced musty odor compounds at 30°C, and differences were observed in musty odor production between various temperatures. This result indicated the potential for odor episodes in water bodies of the tropical countries of Southeast Asia throughout the year due to the mean annual ambient temperature of 27°C in the lowlands.

Effects of Environmental Factors on Cyanobacterial Production of Odorous Compounds: Geosmin and 2-Methylisoborneol.

Geosmin and 2-methylisoborneol (2-MIB), responsible for earthy or musty smell, are a major concern for safe drinking water supplies. This study investigated the effects of environmental factors on odorous compound production and cell growth in cyanobacterial strains. Anabaena sp. FACHB-1384, a 2-MIB producer, was sensitive to low temperature (<20°C). However, geosmin producers, Anabaena sp. Chusori and Anabaena sp. NIER, were sensitive to high light intensity (>100 µmol/m2/sec), but not to low temperature. Geosmin concentrations increased under higher nitrate concentrations, being linearly proportional to cell density. A P-limited chemostat showed that P-stress decreased the geosmin productivity and extracellular geosmin amount per cell in Anabaena sp. NIER. However, only 2-MIB productivity was reduced in Planktothrix sp. FACHB-1374 under P-limitation. The extracellular 2-MIB amount per cell remained constant at all dilution rates. In conclusion, high light intensity and P-stress can contribute to the lower incidence of geosmin, whereas 2-MIB reduction could be attainable at a lower temperature.

Enhancing both oral bioavailability and brain penetration of puerarin using borneol in combination with preparation technologies.

Now there are few good oral preparations of puerarin used in cerebrovascular diseases because of its poor oral absorption caused by the low water solubility and the poor penetration into brain. In this study, three oral formulations of puerarin, nanocrystals suspension (NCS), inclusion compounds solution (ICS) and self-microemulsifying drug delivery system (SMEDDS) were prepared with borneol as an oral brain-targeting enhancer. A rat syngeneic in vitro model of the brain-blood barrier (BBB) was established to investigate effects of borneol on the permeability of puerarin across the BBB. The pharmacokinetics of puerarin in mice after oral administration was investigated by a high performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) method. The in vitro BBB model study showed the permeability of puerarin was increased significantly (p < 0.05) and the value of transepithelial electrical resistance at 2 h was decreased significantly (p < 0.01) when the concentration of borneol was over 12.5 µg/mL compared with the control group. The pharmacokinetics results indicated borneol with doses of over 50 mg/kg could obviously increase both intestinal absorption and brain penetration of puerarin. With co-administration of borneol (100 mg/kg), the AUC of puerarin both in plasma (AUCplasma) and in brain (AUCbrain) for SMEDDS were significantly higher than those for NCS (p < 0.01) and ICS (p < 0.05). These results suggested borneol in combination with SMEDDS could improve both the oral absorption and the brain penetration of puerarin in mice, which was promising for the development of an oral formulation of puerarin used in cerebrovascular diseases.

An alternative method to quantify 2-MIB producing cyanobacteria in drinking water reservoirs: Method development and field applications.

2-Methylisoborneol (2-MIB) is a commonly detected cyanobacterial odorant in drinking water sources in many countries. To provide safe and high-quality water, development of a monitoring method for the detection of 2-MIB-synthesis (mibC) genes is very important. In this study, new primers MIBS02F/R intended specifically for the mibC gene were developed and tested. Experimental results show that the MIBS02F/R primer set was able to capture 13 2-MIB producing cyanobacterial strains grown in the laboratory, and to effectively amplify the targeted DNA region from 17 2-MIB-producing cyanobacterial strains listed in the literature. The primers were further coupled with a TaqMan probe to detect 2-MIB producers in 29 drinking water reservoirs (DWRs). The results showed statistically significant correlations between mibC genes and 2-MIB concentrations for the data from each reservoir (R2=0.413-0.998; p<0.05), from all reservoirs in each of the three islands (R2=0.302-0.796; p<0.01), and from all data of the three islands (R2=0.473-0.479; p<0.01). The results demonstrate that the real-time PCR can be an alternative method to provide information to managers of reservoirs and water utilities facing 2-MIB-related incidents.