Probing Downstream Olive Biophenol Secoiridoids.

Numerous bioactive biophenol secoiridoids (BPsecos) are found in the fruit, leaves, and oil of olives. These BPsecos play important roles in both the taste of food and human health. The main BPseco bioactive from green olive fruits, leaves, and table olives is oleuropein, while olive oil is rich in oleuropein downstream pathway molecules. The aim of this study was to probe olive BPseco downstream molecular pathways that are alike in biological and olive processing systems at different pHs and reaction times. The downstream molecular pathway were analyzed by high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI/MS) and typed neglected of different overlap (TNDO) computational methods. Our study showed oleuropein highest occupied molecular orbital (HOMO) and HOMO-1 triggered the free radical processes, while HOMO-2 and lowest unoccupied molecular orbital (LUMO) were polar reactions of glucoside and ester groups. Olive BPsecos were found to be stable under acid and base catalylic experiments. Oleuropein aglycone opened to diales and rearranged to hydroxytyrosil-elenolate under strong reaction conditions. The results suggest that competition among olive BPseco HOMOs could induce glucoside hydrolysis during olive milling due to native olive ß-glucosidases. The underlined olive BPsecos downstream molecular mechanism herein could provide new insights into the olive milling process to improve BPseco bioactives in olive oil and table olives, which would enhance both the functional food and the nutraceuticals that are produced from olives.

Upstream biomanufacturing of pharmaceutical colchicine.

The plant-based colchicine potentially affects major diseases such as cardiovascular events, cancers and gout. Gloriosa superba seeds are a conventional pharmaceutical source of colchicine. The demand for pharmaceutical-grade colchicine has increased due to the shortage of feasible upstream manufacturing, encompassing all stages in the processes of biosynthesis and biomanufacturing before the raw material is ready for purification. Consequently, developing sustainable upstream industrial colchicine biofactories is imperative, especially in curtailing drug costs. A new upstream bioprocess has been established, using specialized biorhizomes with comprehensive specific-enzymes that catalyze the construction of biogenic functionalized intermediates that are converted into colchicine. This review emphasizes a novel biorhizome approach for biomanufacturing pharmaceutical-grade natural colchicine, a biosynthetic pathway elucidation and its challenges to synthetic biotechnology.

Oleuropein: Molecular Dynamics and Computation.

BACKGROUND: Olive oil and table olive biophenols have been shown to significantly enrich the hedonic-sensory and nutritional quality of the Mediterranean diet. Oleuropein is one of the predominant biophenols in green olives and leaves, which not only has noteworthy freeradical quenching activity but also putatively reduces the incidence of various cancers. Clinical trials suggest that the consumption of extra virgin olive oil reduces the risk of several degenerative diseases. The oleuropein-based bioactives in olive oil could reduce tumor necrosis factor α, interleukin-1ß and nitric oxide. Therefore, the quality of olive biophenols should be preserved and even improved due to their disease-fighting properties. OBJECTIVE: Understanding the molecular dynamics of oleuropein is crucial to increase olive oil and table olive quality. The objective of this review is to provide the molecular dynamics and computational mapping of oleuropein. METHOD: The oleuropein molecular bond sequential breaking mechanisms were analyzed through unimolecular reactions under electron spray ionization, collision activated dissociations, and fast atom bombardment mass spectrometry. RESULTS: Oleuropein is a biophenol-secoiridoid expressing different functionalities such as two π-bonds, two esters, two acetals, one catechol, and four hexose hydroxyls within 540 mw. The oleuropein solvent-free reactivity is leading to glucose loss and bioactive aglycone-dialdehydes via secoiridoid ring opening. CONCLUSION: Oleuropein electron distribution revealed that the free-radical non-polar processes occur from its highest occupied molecular orbital, while the lowest unoccupied molecular orbital is clearly devoted to nucleophilic and base site reactivity. This molecular dynamics and computational mapping of oleuropein could contribute to the engineering of olive-based biomedicine and/or functional food.

Biorhizome: A Biosynthetic Platform for Colchicine Biomanufacturing.

Colchicine is one of the oldest plant-based medicines used to treat gout and one of the most important alkaloid-based antimitotic drugs with anticancer potential, which is commercially extracted from Gloriosa superba. Clinical trials suggest that colchicine medication could prevent atrial fibrillation recurrence after cardiac surgery. In addition, therapeutic colchicine is undergoing clinical trials to treat non-diabetic metabolic syndrome and diabetic nephropathy. However, the industrial-scale biomanufacturing of colchicine have not yet been established. Clearly, further studies on detailed biorhizome-specific transcriptome analysis, gene expression, and candidate gene validation are required before uncover the mechanism of colchicine biosynthesis and biorhizome-based colchicine biomanufacturing. Annotation of 32312 assembled multiple-tissues transcripts of G. superba represented 15088 unigenes in known plant specific gene ontology. This could help understanding colchicine biosynthesis in G. superba. This review highlights the biorhizomes, rhizome specific genes or gene what expressed with high level in rhizomes, and deep fluid dynamics in a bioreactor specifically for the biomanufacture of colchicine.

Soft-MS and Computational Mapping of Oleuropein.

Olive oil and table olives are rich sources of biophenols, which provides a unique taste, aroma and potential health benefits. Specifically, green olive drupes are enriched with oleuropein, a bioactive biophenol secoiridoid. Olive oil contains hydrolytic derivatives such as hydroxytyrosol, oleacein and elenolate from oleuropein as well as tyrosol and oleocanthal from ligstroside. Biophenol secoiridoids are categorized by the presence of elenoic acid or its derivatives in their molecular structure. Medical studies suggest that olive biophenol secoiridoids could prevent cancer, obesity, osteoporosis, and neurodegeneration. Therefore, understanding the biomolecular dynamics of oleuropein can potentially improve olive-based functional foods and nutraceuticals. This review provides a critical assessment of oleuropein biomolecular mechanism and computational mapping that could contribute to nutrigenomics.

Biomass and RRR-α-tocopherol production in Stichococcus bacillaris strain siva2011 in a balloon bioreactor.

BACKGROUND: Green microalgae represent a renewable natural source of vitamin E. Its most bioactive form is the naturally occurring RRR-α-tocopherol which is biosynthesized in photosynthetic organisms as a single stereoisomer. It is noteworthy that the natural and synthetic α-tocopherols are different biomolecular entities. This article focuses on RRR-α-tocopherol production in Stichococcus bacillaris strain siva2011 biomass in a bioreactor culture with methyl jasmonate (MeJa) elicitor. Additionally, a nonlinear mathematical model was used to quantitatively scale-up and predict the biomass production in a 20 L balloon bioreactor with dual variables such as time and volume. RESULTS: Approximately 0.6 mg/g dry weight (DW) of RRR-α-tocopherol was enhanced in S. bacillaris strain siva2011 biomass with the MeJa 50 µL/L for 24 hrs elicitations when compared to the control. The R2 value from the nonlinear model was enhanced up to 95% when compared to the linear model which significantly improved the accuracy for estimating S. bacillaris strain siva2011 biomass production in a balloon bioreactor. CONCLUSIONS: S. bacillaris strain siva2011 is a new green microalga which biosynthesizes significant amounts of RRR-α-tocopherol. Systematically validated dual variable empirical data should provide key insights to multivariable or fourth order modeling for algal biomass scale-up. This bioprocess engineering should provide valuable information for industrial production of RRR-α-tocopherol from green cells.

Bioprocessing of Stichococcus bacillaris strain siva2011.

BACKGROUND: Globally, the development of a cost-effective long-term renewable energy infrastructure is one of the most challenging problems faced by society today. Microalgae are rich in potential biofuel substrates such as lipids, including triacylglycerols (TAGs). Some of these algae also biosynthesize small molecule hydrocarbons. These hydrocarbons can often be used as liquid fuels, often with more versatility and by a more direct approach than some TAGs. However, the appropriate TAGs, accumulated from microalgae biomass, can be used as substrates for different kinds of renewable liquid fuels such as biodiesel and jet fuel. RESULTS: This article describes the isolation and identification of a lipid-rich, hydrocarbon-producing alga, Stichococcus bacillaris strain siva2011, together with its bioprocessing, hydrocarbon and fatty acid methyl ester (FAME) profiles. The S. bacillaris strain siva2011 was scaled-up in an 8 L bioreactor with 0.2% CO2. The C16:0, C16:3, C18:1, C18:2 and C18:3 were 112.2, 9.4, 51.3, 74.1 and 69.2 mg/g dry weight (DW), respectively. This new strain produced a significant amount of biomass of 3.79 g/L DW on day 6 in the 8 L bioreactor and also produced three hydrocarbons. CONCLUSIONS: A new oil-rich microalga S. bacillaris strain siva2011 was discovered and its biomass has been scaled-up in a newly designed balloon-type bioreactor. The TAGs and hydrocarbons produced by this organism could be used as substrates for jet fuel or biodiesel.

Integrated green algal technology for bioremediation and biofuel.

Sustainable non-food energy biomass and cost-effective ways to produce renewable energy technologies from this biomass are continuously emerging. Algae are capable of producing lipids and hydrocarbons quickly and their photosynthetic abilities make them a promising candidate for an alternative energy source. In addition, their favorable carbon life cycle and a renewed focus on rural economic development are attractive factors. In this review the focus is mainly on the integrated approach of algae culture for bioremediation and oil-based biofuel production with mention of possible other value-added benefits of using algae for those purposes.

Bioethanol production from dedicated energy crops and residues in Arkansas, USA.

Globally, one of the major technologic goals is to achieve cost-effective lignocellulosic ethanol production from biomass feedstocks. Lignocellulosic biomass of four dedicated energy crops [giant reed (Arundo donax L.), elephantgrass (Pennisetum purpureum (Schumach), Miscanthus × giganteus (Illinois clone), and (clone Q42641) {hybrid of Miscanthus sinensis Anderss. and Miscanthus sacchariflorus (Maxim)}, Hack. called giant miscanthus, and sugarcane clone US 84-1028 (Saccharum L. spp. hybrid)] and residues from two crops [soybean (Glycine max (L.) Merr.) litter and rice (Oryza sativa L.) husk] were tested for bioethanol production using cellulose solvent-based lignocellulose fractionation (CSLF) pretreatment and enzymatic (cellulase) hydrolysis. Giant miscanthus (Illinois), giant reed, giant miscanthus (Q42641), elephantgrass, and sugarcane all yielded higher amount of glucose on a biomass dry weight basis (0.290-0.331 g/g), than did rice husk (0.181 g/g) and soybean litter (0.186 g/g). To reduce the capital investment for energy consumption in fermentation, we used a self-flocculating yeast strain (SPSC01) to ferment the lignocellulosic biomass hydrolysates. Bioethanol production was ∼0.1 g/g in dedicated energy crops and less in two crop residues. These methods and data can help to develop a cost-effective downstream process for bioethanol production.

Haptic controlled three-axis MEMS gripper system.

In this work, we describe the development and testing of a three degree of freedom meso/micromanipulation system for handling micro-objects, including biological cells and microbeads. Three-axis control is obtained using stepper motors coupled to micromanipulators. The test specimen is placed on a linear X-stage, which is coupled to one stepper motor. The remaining two stepper motors are coupled to the Y and Z axes of a micromanipulator. The stepper motor-micromanipulator arrangement in the Y and Z axes has a minimum step resolution of ∼0.4 µm with a total travel of 12 mm and the stepper motor-X stage arrangement has a minimum resolution of ∼0.3 µm with a total travel of 10 mm. Mechanical backlash error is ∼0.8 µm for ∼750 µm of travel. A MEMS microgripper from Femtotools™ acts as an end-effector in the shaft end of the micromanipulator. The gripping ranges of the grippers used are 0-100 µm (for FT-G100) and 0-60 µm (for FT-G60). As the gripping action is performed, the force sense circuit of FT-G100 measures the handling force. This force feedback is integrated to a commercially available three degree of freedom haptic device (Novint Falcon) allowing the user to receive tactile feedback during the microscale handling. Both mesoscale and microscale controls are important, as mesoscale control is required for the travel motion of the test object whereas microscale control is required for the gripping action. The haptic device is used to control the position of the microgripper, control the actuation of the microgripper, and provide force feedback. A LABVIEW program was developed to interlink communication and control among hardware used in the system. Micro-objects such as SF-9 cells and polystyrene beads (∼45 µm) are handled and handling forces of ∼50 µN were experienced.

Biomass production of hairy roots of Artemisia annua and Arachis hypogaea in a scaled-up mist bioreactor.

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low-cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2-3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2-3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day(-1) with biomass yield of 12.75 g DW L(-1). This exceeded that in shake flasks at µ = 0.166 day(-1) and 11.10 g DW L(-1). Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L(-1), which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots.

Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: Effects of culture medium and growth stage.

Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds.

Plant-based corosolic acid: future anti-diabetic drug?

Diabetes is one of the nation's most prevalent, debilitating and costly diseases. For diabetes, frequent insulin treatment is very expensive and may increase anti-insulin antibody production, which may cause unwanted side effects. Corosolic acid may also have some efficacy in the treatment of diabetes, but without induction of anti-insulin antibodies. Recently, corosolic acid from Lagerstroemia speciosa L. leaf extracts has been reported to act via an indirect mechanism (unlike insulin) in animal experiments. The insulin-complementary anti-diabetic therapeutic value observed in these Japanese preliminary clinical trials has led to renewed interest in the biosynthesis of this compound. So far, there has been no clear evidence for a corosolic acid biosynthetic pathway in plants. This article provides possible roles of corosolic acid and hypothetical information on the biosynthetic pathway in plants.

Functional characterization of a stilbene synthase gene using a transient expression system in planta.

The expression and functionality of a resveratrol synthase (RS) gene from peanut (Arachis hypogaea) was studied using an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana leaves. Functional analysis of RS was demonstrated by tracking its expression during 96 h. To measure the transcripts levels of RS transgene, real-time qRT-PCR was used and revealed that the highest level of transcripts was at 48 h post-transfection. Western blot analyses showed that RS protein was accumulated to the highest levels at 72 h post-transfection. Finally, HPLC and mass spectrometry analyses revealed the production of trans-piceid (resveratrol glucoside) as the major stilbenoid compound confirming the functional activity of the RS enzyme in planta. No activity of RS transgene was detected in negative controls. This strategy showed advantages over conventional systems because it does not require establishment of cell cultures, feeding with appropriate substrates or generation of stable transgenic plants. This transient system proved to be a rapid and direct approach to perform functional analysis of stilbene synthases, such as resveratrol synthase. Furthermore, this approach can be useful to study the metabolic effects of over-expressing or silencing specific genes within a short period of time.

Demethyloleuropein and beta-glucosidase activity in olive fruits.

Demethyloleuropein plays a major role in the defense mechanism of olive fruits. To understand how this molecule is metabolized during different stages of maturation of olive fruits, a biomolecular approach to identify the demethyloleuropein chemistry was employed. The beta-glucosidase activity in crude extracts was assayed spectrophotometrically using the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Demethyloleuropein was extracted and identified by HPLC-MS from both infected and uninfected olive fruits at different physiological stages. The release of more functionally relevant dialdehydes in uninfected fruits was investigated using ESIMS/ MS. In fruits harvested in October, the activity of beta-glucosidase was significantly enhanced in uninfected fruits when compared to the infected fruits. Quantitative differences in the demethyloleuropein content from uninfected fruits showed the highest values (5.09 mg/g) in October, whereas lower levels (4.44 mg/g) were found in infected fruits. The results demonstrated that demethyloleuropein derivatives could be influenced by beta-glucosidase activity to improve the quality of the olive products with the best dialdehyde nutraceutical content.

Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots.

Plants are the richest source for different bioactive molecules. Because of the vast number of side effects associated with synthetic pharmaceuticals, medical biotechnologists turned to nature to provide new promising therapeutic molecules from plant biofactories. The large-scale availability of the disease- and pesticide-free raw material is, however, restricted in vivo. Many bioactive plant secondary metabolites are accumulated in roots. Engineered plants can also produce human therapeutic proteins. Vaccines and diagnostic monoclonal antibodies can be won from their roots, so that engineered plants hold immense potential for the biopharmaceutical industry. To obtain sufficient amounts of the plant bioactive molecules for application in human therapy, adventitious and hairy roots have to be cultured in in vitro systems. High-tech pilot-scale bioreactor technology for the establishment of a long-term adventitious root culture from biopharmaceutical plants has recently been established. In this review, I briefly discuss a technology for cultivating bioactive molecule-rich adventitious and hairy roots from plants using a high-tech bioreactor system, as well as the principles and application of genome-restructuring mechanisms for plant-based biopharmaceutical production from roots. High-tech bioreactor-derived bioactive phytomolecules and biopharmaceuticals hold the prospect of providing permanent remedies for improving human well-being.