An Engineered Plant Metabolic Pathway Results in High Yields of Hydroxytyrosol Due to a Modified Whole-Cell Biocatalysis in Bioreactor.

Hydroxytyrosol (HT) is a phenolic substance primarily present in olive leaves and olive oil. Numerous studies have shown its advantages for human health, making HT a potentially active natural component with significant added value. Determining strategies for its low-cost manufacturing by metabolic engineering in microbial factories is hence still of interest. The objective of our study was to assess and improve HT production in a one-liter bioreactor utilizing genetically modified Escherichia coli strains that had previously undergone fed-batch testing. Firstly, we compared the induction temperatures in small-scale whole-cell biocatalysis studies and then examined the optimal temperature in a large volume bioreactor. By lowering the induction temperature, we were able to double the yield of HT produced thereby, reaching 82% when utilizing tyrosine or L-DOPA as substrates. Hence, without the need to further modify our original strains, we were able to increase the HT yield.

Hydroxytyrosol produced by engineered Escherichia coli strains activates Nrf2/HO-1 pathway: An in vitro and in vivo study.

This study explores the biological effects of hydroxytyrosol (HT), produced by the metabolic engineering of Escherichia coli, in a series of in vitro and in vivo experiments. In particular, a metabolically engineered Escherichia coli strain capable of producing HT was constructed and utilized. HEK293 and HeLa cells were exposed to purified HT to determine non-toxic doses that can offer protection against oxidative stress (activation of Nrf2/HO-1 signaling pathway). Male CD-1 mice were orally supplemented with HT to evaluate (1) renal and hepatic toxicity, (2) endogenous system antioxidant response, and (3) activation of Nrf2/HO-1 system in the liver. HT protected cells from oxidative stress through the activation of Nrf2 regulatory network. Activation of Nrf2 signaling pathway was also observed in the hepatic tissue of the mice. HT supplementation was safe and produced differential effects on mice's endogenous antioxidant defense system. HT biosynthesized from genetically modified Escherichia coli strains is an alternative method to produce high-quality HT that exerts favorable effects in the regulation of the organism's response to oxidative stress. Nonetheless, further investigation of the multifactorial action of HT on the antioxidant network regulation is needed.

Hybrid Autofluorescence and Optoacoustic Microscopy for the Label-Free, Early and Rapid Detection of Pathogenic Infections in Vegetative Tissues.

Agriculture plays a pivotal role in food security and food security is challenged by pests and pathogens. Due to these challenges, the yields and quality of agricultural production are reduced and, in response, restrictions in the trade of plant products are applied. Governments have collaborated to establish robust phytosanitary measures, promote disease surveillance, and invest in research and development to mitigate the impact on food security. Classic as well as modernized tools for disease diagnosis and pathogen surveillance do exist, but most of these are time-consuming, laborious, or are less sensitive. To that end, we propose the innovative application of a hybrid imaging approach through the combination of confocal fluorescence and optoacoustic imaging microscopy. This has allowed us to non-destructively detect the physiological changes that occur in plant tissues as a result of a pathogen-induced interaction well before visual symptoms occur. When broccoli leaves were artificially infected with Xanthomonas campestris pv. campestris (Xcc), eventually causing an economically important bacterial disease, the induced optical absorption alterations could be detected at very early stages of infection. Therefore, this innovative microscopy approach was positively utilized to detect the disease caused by a plant pathogen, showing that it can also be employed to detect quarantine pathogens such as Xylella fastidiosa.

Vitis vinifera genotyping toolbox to highlight diversity and germplasm identification.

The contribution of vine cultivation to human welfare as well as the stimulation of basic social and cultural features of civilization has been great. The wide temporal and regional distribution created a wide array of genetic variants that have been used as propagating material to promote cultivation. Information on the origin and relationships among cultivars is of great interest from a phylogenetics and biotechnology perspective. Fingerprinting and exploration of the complicated genetic background of varieties may contribute to future breeding programs. In this review, we present the most frequently used molecular markers, which have been used on Vitis germplasm. We discuss the scientific progress that led to the new strategies being implemented utilizing state-of-the-art next generation sequencing technologies. Additionally, we attempted to delimit the discussion on the algorithms used in phylogenetic analyses and differentiation of grape varieties. Lastly, the contribution of epigenetics is highlighted to tackle future roadmaps for breeding and exploitation of Vitis germplasm. The latter will remain in the top of the edge for future breeding and cultivation and the molecular tools presented herein, will serve as a reference point in the challenging years to come.

Oral Supplementation with Hydroxytyrosol Synthesized Using Genetically Modified Escherichia coli Strains and Essential Oils Mixture: A Pilot Study on the Safety and Biological Activity.

Several natural compounds have been explored as immune-boosting, antioxidant and anti-inflammatory dietary supplements. Amongst them, hydroxytyrosol, a natural antioxidant found in olive products, and endemic medicinal plants have attracted the scientific community's and industry's interest. We investigated the safety and biological activity of a standardised supplement containing 10 mg of hydroxytyrosol synthesized using genetically modified Escherichia coli strains and equal amounts (8.33 µL) of essential oils from Origanum vulgare subsp. hirtum, Salvia fruticosa and Crithmum maritimum in an open-label, single-arm, prospective clinical study. The supplement was given to 12 healthy subjects, aged 26-52, once a day for 8 weeks. Fasting blood was collected at three-time points (weeks 0, 8 and follow-up at 12) for analysis, which included full blood count and biochemical determination of lipid profile, glucose homeostasis and liver function panel. Specific biomarkers, namely homocysteine, oxLDL, catalase and total glutathione (GSH) were also studied. The supplement induced a significant reduction in glucose, homocysteine and oxLDL levels and was tolerated by the subjects who reported no side effects. Cholesterol, triglyceride levels and liver enzymes remained unaffected except for LDH. These data indicate the supplement's safety and its potential health-beneficial effects against pathologic conditions linked to cardiovascular disease.

Evolution of Physicochemical Properties and Phenolic Maturity of Vilana, Vidiano, Kotsifali and Mandilari Wine Grape Cultivars (Vitis vinifera L.) during Ripening.

Determining the optimum harvest time is a significant factor affecting the quality of the grapes and the wine. Monitoring the evolution of grapes' physicochemical properties and phenolic maturity during ripening could be a valuable tool for determining the optimum harvest time. In this study, the total phenolic content, antioxidant activity, flavonols, flavanols, anthocyanins and resveratrol content were determined during the last weeks of ripening for the white cultivars Vilana and Vidiano, as well as for the red cultivars Kotsifali and Mandilari (Vitis vinifera L.). According to the results, an early harvest for the white cultivars and a late harvest for the red cultivars may increase the total phenolics and trans-resveratrol content in grapes and wine. An early harvest would be desirable to maintain high flavanols content and high levels of antioxidant activity in the grapes' skin and seeds. Conversely, a late harvest for the red cultivars may be desirable to increase the total flavonols and anthocyanin content in grapes and wines.

Resveratrol: A Fair Race Towards Replacing Sulfites in Wines.

In recent years, significant efforts to produce healthier wines has led to the replacement or reduction of the addition of sulfites, using alternative substances or techniques. Resveratrol and related biophenols seem to be of great interest, since beyond their protective nature and contrary to sulfites they can positively affect consumer health. These bioactive phytochemicals are naturally produced in grapes as evolutionary acquired mechanisms against pathogens and UV irradiation. However, despite the efforts made so far attempting to develop economic and industrially adopted isolation techniques, available quantities of these biophenols for commercial use are still quite limited. Therefore, such molecules are still not able to meet the needs of industrial use due to their prohibitive marketable cost. In this review we summarize the efforts that have been made to biosynthesize these molecules through alternative, innovative ways. Increasing interest in modern biotechnological approaches has shed light on the exploitation of metabolically engineered microbial factories, instead of plants, to produce molecules of industrial interest. Such approaches, also reviewed here, are expected to lower the cost and appear promising to produce enough surplus to attract further oenological experimentation upon yielding functional wines. This development is expected to attract further industrial attention, continuing the race to partially or totally replace the external addition of sulfites. We also review important physicochemical properties of resveratrol in relation to enriching wines.

Dual pathway for metabolic engineering of Escherichia coli to produce the highly valuable hydroxytyrosol.

One of the most abundant phenolic compounds traced in olive tissues is hydroxytyrosol (HT), a molecule that has been attributed with a pile of beneficial effects, well documented by many epidemiological studies and thus adding value to products containing it. Strong antioxidant capacity and protection from cancer are only some of its exceptional features making it ideal as a potential supplement or preservative to be employed in the nutraceutical, agrochemical, cosmeceutical, and food industry. The HT biosynthetic pathway in plants (e.g. olive fruit tissues) is not well apprehended yet. In this contribution we employed a metabolic engineering strategy by constructing a dual pathway introduced in Escherichia coli and proofing its significant functionality leading it to produce HT. Our primary target was to investigate whether such a metabolic engineering approach could benefit the metabolic flow of tyrosine introduced to the conceived dual pathway, leading to the maximalization of the HT productivity. Various gene combinations derived from plants or bacteria were used to form a newly inspired, artificial biosynthetic dual pathway managing to redirect the carbon flow towards the production of HT directly from glucose. Various biosynthetic bottlenecks faced due to feaB gene function, resolved through the overexpression of a functional aldehyde reductase. Currently, we have achieved equimolar concentration of HT to tyrosine as precursor when overproduced straight from glucose, reaching the level of 1.76 mM (270.8 mg/L) analyzed by LC-HRMS. This work realizes the existing bottlenecks of the metabolic engineering process that was dependent on the utilized host strain, growth medium as well as to other factors studied in this work.

The Interplay among Polyamines and Nitrogen in Plant Stress Responses.

The interplay between polyamines (PAs) and nitrogen (N) is emerging as a key factor in plant response to abiotic and biotic stresses. The PA/N interplay in plants connects N metabolism, carbon (C) fixation, and secondary metabolism pathways. Glutamate, a pivotal N-containing molecule, is responsible for the biosynthesis of proline (Pro), arginine (Arg) and ornithine (Orn) and constitutes a main common pathway for PAs and C/N assimilation/incorporation implicated in various stresses. PAs and their derivatives are important signaling molecules, as they act largely by protecting and preserving the function/structure of cells in response to stresses. Use of different research approaches, such as generation of transgenic plants with modified intracellular N and PA homeostasis, has helped to elucidate a plethora of PA roles, underpinning their function as a major player in plant stress responses. In this context, a range of transgenic plants over-or under-expressing N/PA metabolic genes has been developed in an effort to decipher their implication in stress signaling. The current review describes how N and PAs regulate plant growth and facilitate crop acclimatization to adverse environments in an attempt to further elucidate the N-PAs interplay against abiotic and biotic stresses, as well as the mechanisms controlling N-PA genes/enzymes and metabolites.

Microalgal biofilms: A further step over current microalgal cultivation techniques.

The scientific community has turned its interest to microalgae lately, because of their countless applications such as wastewater treatment and pharmaceutical industry. Nevertheless, so far applied cultivation methods are still prohibitive. Ordinary cultivation techniques in which microalgae are suspended in liquid medium suffer from many bottlenecks, such as low biomass productivities, difficulty in biomass harvesting and recovery, high installation and operating cost, high water requirements etc. Although, microalgal biofilms are known to be a nuisance because of surfaces fouling, they have emerged as an innovative technology with which microalgae are developed attached to a solid surface. This technique seems to be advantageous as compared to conventional cultivation systems. Microalgal biofilm systems could resolve the problematic aspects of ordinary cultivation techniques such as low biomass productivities, water management and biomass recovery. A detailed description of this technique with respect to the parameters affecting them is reviewed in this work.

Expression of hydroxytyrosol and oleuropein biosynthetic genes are correlated with metabolite accumulation during fruit development in olive, Olea europaea, cv. Koroneiki.

Olive tree is one of the most valuable crops cultivated for its oil that is rich in antioxidants. The beneficial effects of oleuropein and hydroxytyrosol (HT), the most abundant and the most powerful antioxidant respectively, as well as tyrosol, HT's precursor molecule, are well studied however their biosynthetic pathways are not yet clarified. The transcriptome analysis of the young olive fruit, cultivar "Koroneiki", revealed transcripts of all the enzymes used to reconstitute the biosynthetic pathway of tyrosol and HT in other organisms. We also identified transcripts of the genes that encode for enzymes involved in the secologanin biosynthesis, oleuropein's precursor molecule. Following the transcriptome analysis, the relative expression of the transcripts was monitored during fruit development and compared to the concentration of the 3 metabolites they synthesize at the same developmental stages. The highest expression levels, accompanied by the maximum concentration of the three metabolites, was found in the young olive fruit. The correlation between the expression profile and the metabolites' concentration indicates that the transcripts were correctly identified and the synthesis of the compounds is regulated at a transcriptional level. Interestingly, HT showed a sudden increment in the final developmental stage of the black mature fruit that is attributed to oleuropein catabolism.

Sensitive cells: enabling tools for static and dynamic control of microbial metabolic pathways.

Natural metabolic pathways are dynamically regulated at the transcriptional, translational, and protein levels. Despite this, traditional pathway engineering has relied on static control strategies to engender changes in metabolism, most likely due to ease of implementation and perceived predictability of design outcome. Increasingly in recent years, however, metabolic engineers have drawn inspiration from natural systems and have begun to harness dynamically controlled regulatory machinery to improve design of engineered microorganisms for production of specialty and commodity chemicals. Here, we review recent enabling technologies for engineering static control over pathway expression levels, and we discuss state-of-the-art dynamic control strategies that have yielded improved outcomes in the field of microbial metabolic engineering. Furthermore, we emphasize design of a novel class of genetically encoded controllers that will facilitate automatic, transient tuning of synthetic and endogenous pathways.

Comparative genomic analysis of multiple strains of two unusual plant pathogens: Pseudomonas corrugata and Pseudomonas mediterranea.

The non-fluorescent pseudomonads, Pseudomonas corrugata (Pcor) and P. mediterranea (Pmed), are closely related species that cause pith necrosis, a disease of tomato that causes severe crop losses. However, they also show strong antagonistic effects against economically important pathogens, demonstrating their potential for utilization as biological control agents. In addition, their metabolic versatility makes them attractive for the production of commercial biomolecules and bioremediation. An extensive comparative genomics study is required to dissect the mechanisms that Pcor and Pmed employ to cause disease, prevent disease caused by other pathogens, and to mine their genomes for genes that encode proteins involved in commercially important chemical pathways. Here, we present the draft genomes of nine Pcor and Pmed strains from different geographical locations. This analysis covered significant genetic heterogeneity and allowed in-depth genomic comparison. All examined strains were able to trigger symptoms in tomato plants but not all induced a hypersensitive-like response in Nicotiana benthamiana. Genome-mining revealed the absence of type III secretion system and known type III effector-encoding genes from all examined Pcor and Pmed strains. The lack of a type III secretion system appears to be unique among the plant pathogenic pseudomonads. Several gene clusters coding for type VI secretion system were detected in all genomes. Genome-mining also revealed the presence of gene clusters for biosynthesis of siderophores, polyketides, non-ribosomal peptides, and hydrogen cyanide. A highly conserved quorum sensing system was detected in all strains, although species specific differences were observed. Our study provides the basis for in-depth investigations regarding the molecular mechanisms underlying virulence strategies in the battle between plants and microbes.

When plants produce not enough or at all: metabolic engineering of flavonoids in microbial hosts.

As a result of the discovery that flavonoids are directly or indirectly connected to health, flavonoid metabolism and its fascinating molecules that are natural products in plants, have attracted the attention of both the industry and researchers involved in plant science, nutrition, bio/chemistry, chemical bioengineering, pharmacy, medicine, etc. Subsequently, in the past few years, flavonoids became a top story in the pharmaceutical industry, which is continually seeking novel ways to produce safe and efficient drugs. Microbial cell cultures can act as workhorse bio-factories by offering their metabolic machinery for the purpose of optimizing the conditions and increasing the productivity of a selective flavonoid. Furthermore, metabolic engineering methodology is used to reinforce what nature does best by correcting the inadequacies and dead-ends of a metabolic pathway. Combinatorial biosynthesis techniques led to the discovery of novel ways of producing natural and even unnatural plant flavonoids, while, in addition, metabolic engineering provided the industry with the opportunity to invest in synthetic biology in order to overcome the currently existing restricted diversification and productivity issues in synthetic chemistry protocols. In this review, is presented an update on the rationalized approaches to the production of natural or unnatural flavonoids through biotechnology, analyzing the significance of combinatorial biosynthesis of agricultural/pharmaceutical compounds produced in heterologous organisms. Also mentioned are strategies and achievements that have so far thrived in the area of synthetic biology, with an emphasis on metabolic engineering targeting the cellular optimization of microorganisms and plants that produce flavonoids, while stressing the advances in flux dynamic control and optimization. Finally, the involvement of the rapidly increasing numbers of assembled genomes that contribute to the gene- or pathway-mining in order to identify the gene(s) responsible for producing species-specific secondary metabolites is also considered herein.

Comparative genomics of multiple strains of Pseudomonas cannabina pv. alisalensis, a potential model pathogen of both monocots and dicots.

Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.

Pseudomonas viridiflava, a multi host plant pathogen with significant genetic variation at the molecular level.

The pectinolytic species Pseudomonas viridiflava has a wide host range among plants, causing foliar and stem necrotic lesions and basal stem and root rots. However, little is known about the molecular evolution of this species. In this study we investigated the intraspecies genetic variation of P. viridiflava amongst local (Cretan), as well as international isolates of the pathogen. The genetic and phenotypic variability were investigated by molecular fingerprinting (rep-PCR) and partial sequencing of three housekeeping genes (gyrB, rpoD and rpoB), and by biochemical and pathogenicity profiling. The biochemical tests and pathogenicity profiling did not reveal any variability among the isolates studied. However, the molecular fingerprinting patterns and housekeeping gene sequences clearly differentiated them. In a broader phylogenetic comparison of housekeeping gene sequences deposited in GenBank, significant genetic variability at the molecular level was found between isolates of P. viridiflava originated from different host species as well as among isolates from the same host. Our results provide a basis for more comprehensive understanding of the biology, sources and shifts in genetic diversity and evolution of P. viridiflava populations and should support the development of molecular identification tools and epidemiological studies in diseases caused by this species.

Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae.

Chemical or biological synthesis of plant secondary metabolites has attracted increasing interest due to their proven or assumed beneficial properties and health promoting effects. Resveratrol, a stilbenoid, naringenin, a flavanone, genistein, an isoflavone, and the flavonols kaempferol and quercetin have been shown to possess high nutritional and agricultural value. Four metabolically engineered yeast strains harboring plasmids with heterologous genes for enzymes involved in the biosynthesis of these compounds from phenylalanine have been constructed. Time course analyses of precursor utilization and end-product accumulation were carried out establishing the production of 0.29-0.31 mg/L of trans-resveratrol, 8.9-15.6 mg/L of naringenin, 0.1-7.7 mg/L of genistein, 0.9-4.6 mg/L of kaempferol and 0.26-0.38 mg/L of quercetin in defined media under optimal growth conditions. The recombinant yeast strains can be used further for the construction of improved flavonoid- and stilbenoid-overproducers.

Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part I: Chemical diversity, impacts on plant biology and human health.

Plant natural products derived from phenylalanine and the phenylpropanoid pathway are impressive in their chemical diversity and are the result of plant evolution, which has selected for the acquisition of large repertoires of pigments, structural and defensive compounds, all derived from a phenylpropanoid backbone via the plant-specific phenylpropanoid pathway. These compounds are important in plant growth, development and responses to environmental stresses and thus can have large impacts on agricultural productivity. While plant-based medicines containing phenylpropanoid-derived active components have long been used by humans, the benefits of specific flavonoids and other phenylpropanoid-derived compounds to human health and their potential for long-term health benefits have been only recognized more recently. In this part of the review, we discuss the diversity and biosynthetic origins of phenylpropanoids and particularly of the flavonoid and stilbenoid natural products. We then review data pertaining to the modes of action and biological properties of these compounds, referring on their effects on human health and physiology and their roles as plant defense and antimicrobial compounds. This review continues in Part II discussing the use of biotechnological tools targeting the rational reconstruction of multienzyme pathways in order to modify the production of such compounds in plants and model microbial systems for the benefit of agriculture and forestry.

Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part II: Reconstruction of multienzyme pathways in plants and microbes.

Plant natural products derived from phenylalanine and the phenylpropanoid pathways are impressive in their chemical diversity and are the result of plant evolution, which has selected for the acquisition of large repertoires of pigments, structural and defensive compounds, all derived from a phenylpropanoid backbone via the plant-specific phenylpropanoid pathway. These compounds are important in plant growth, development and responses to environmental stresses and thus can have large impacts on agricultural productivity. While plant-based medicines containing phenylpropanoid-derived active components have long been used by humans, the benefits of specific flavonoids and other phenylpropanoid-derived compounds to human health and their potential for long-term health benefits have only been recognized more recently. In this part of the review, we discuss in detail the recent strategies and achievements used in the reconstruction of multienzyme pathways in plants and microbes in an effort to be able to attain higher amounts of the desired flavonoids and stilbenoids exploiting their beneficial properties as analyzed extensively in Part I of this review.