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.National Strategic Reference Framework. THALES-TEI CRETE, MIS 380210 Progra

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

Heterologous biosynthesis of plant flavonoids from Saccharomyses serevisiae, targeting their exploitation in phytoprotection of grapevine and wine making

In addition to many essential nutritional components, plants contain (poly)phenolic substances that comprise a large and heterogeneous group of biologically active non-nutrient compounds. Among them flavonoids and stilbenoids are of exceptional interest, with the former consisting of a diverse array of structurally related compounds possessing the same C6-C3-C6 backbone skeleton, derived from the phenylpropanoid biosynthetic pathway. The term “flavonoids” is used to include the three classes of structurally related compounds deriving from the condensation reaction of an activated hydroxy-cinnamic acid (phenylpropanoid) with malonyl-CoA, main flavonoids, isoflavonoids and neoflavonoids. On the other hand stilbenoids are compounds derived by a similar condensation reaction but with the participation of an evolutionarily diverged enzyme. The significance of flavonoids and stilbenoids derives from the fact that they are involved in aspects of plant physiology such as tissue pigmentation, defence against microbes or insects, interactions with other organisms, protection against UV irradiation, transmission and response to specific environmental stimuli, pollen germination and active auxin transfer. Moreover these compounds, as found recently, have also high pharmacological value. Flavonoids have long been recognised to possess anti-oxidant, anti-inflammatory, antimicrobial, anti-proliferative, anti-allergenic activities and acting as post-menopausal and cardiovascular cures. Inverse relationships between the intake of flavonoids and the risk of coronary heart disease, stroke and many types of cancer have been shown by epidemiological studies. A plethora of secondary metabolites with such exceptional properties are produced through plant’s metabolic machinery in traceable quantities or are extremely difficult to extract or process them. The interest for the exploitation of these metabolite properties by the pharmaceutical or agricultural industry (biological or integrated crop protection) and medicine has increased the need for heterologous biosynthesis of substances that are assumed or proven to possess beneficial actions, such as the flavonoids and stilbenoids. Coumaric acid, a phenolic acid, resveratrol, a stilbenoid, naringenin, a flavanone, genistein, an isoflavone, and flavonols kaempferol and quercetin have been shown to be substances with potential nutritional and agricultural value. In this doctoral dissertation, six metabolically engineered yeast (Saccharomyces cerevisiae) strains harbouring plasmids with all the necessary genes that permit the biosynthesis of the abovementioned compounds, utilizing phenylalanine as a precursor, have been constructed. Yeast strains with transcriptionally active heterologous genes, were used to construct time courses showing the dependence of the precursor utilization and the quantification of the respective end-product synthesis. Thus it has been demonstrated the production of 108,6mg/L of coumaric acid, 0,29-0,31mg/L of resveratrol, 8,9-15,6mg/L of naringenin, 0,1-7,7mg/L of genistein, 0,9-4,6mg/L of kaempferol and 0,26-0,38mg/L of quercetin in culture media, with respect to the precursor molecule that has been tested. Moreover, experiments were performed to optimize the parameters involved in the heterologous biosynthetic pathways. Especially, the optimal concentration of the precursor molecule was studied as well as the relevance of starting inoculum at the end-product concentration. Furthermore, the fluxes through the intermediate nodes of the pathways were studied. Finally, the advantages and disadvantages of the use of yeast are discussed as being a host for the heterologous production of flavonoids and stilbenoids and the kind of their potential uses in wine and agricultural (phytoprotection) industry.Οι (πολυ)φαινολικές ουσίες είναι μια σημαντική κατηγορία φυτικών συστατικών που εμπλέκονται στο διατροφικό μενού του ανθρώπου. Οι ουσίες αυτές αποτελούν μια πολυσύστατη ομάδα με βιολογικώς δραστικές μη-θρεπτικές ενώσεις. Μεταξύ αυτών, τα φλαβονοειδή και τα στιλβενοειδή παρουσιάζουν εξαιρετικό ενδιαφέρον, με τα πρώτα να αποτελούνται από ενώσεις με συναφή χημική δομή, με την έννοια ότι στηρίζονται στον ίδιο C6-C3-C6 σκελετό, προερχόμενα από το βιοσυνθετικό μονοπάτι των φαινυλοπροπανοειδών. Ο όρος «φλαβονοειδή» χρησιμοποιείται για να συμπεριλάβει τρεις τάξεις συναφών δομικά ομάδων ενώσεων που απορρέουν από μια αντίδραση συμπύκνωσης ενός «ενεργοποιημένου» υδροξυ-κινναμικού οξέος (φαινυλοπροπανοειδές) με το μηλόνυλο-CoA, τα κύρια φλαβονοειδή, τα ισοφλαβονοειδή και τα νεοφλαβονοειδή. Από την άλλη πλευρά, τα στιλβενοειδή είναι ενώσεις που προέρχονται από μια παρόμοια αντίδραση συμπύκνωσης, αλλά με την διαμεσολάβηση ενός εξελικτικά διαφοροποιημένου ενζύμου. Η εξέχουσα σημασία των φλαβονοειδών και των στιλβενοειδών πηγάζει από το γεγονός ότι εμπλέκονται σε πολλές λειτουργίες της φυσιολογίας των φυτών, όπως τον χρωματισμό των ιστών, την ανθεκτικότητα ενάντια σε μικρόβια και έντομα, τις αλληλεπιδράσεις με άλλους οργανισμούς, την προστασία από την υπεριώδη ακτινοβολία, την μετάδοση και απόκριση σε ερεθίσματα του περιβάλλοντος, την βλάστηση της γύρης και την ενεργό μεταφορά αυξινών. Επιπλέον, τα συστατικά αυτά, όπως αποδείχθηκε πρόσφατα, έχουν εξαιρετικά υψηλή φαρμακολογική αξία. Οι φλαβονοειδείς ενώσεις διαθέτουν αντι- οξειδωτικές, αντι-φλεγμονώδεις, αντι-μικροβιακές, αντι-καρκινικές ιδιότητες όπως επίσης μπορούν να χρησιμοποιηθούν για την καταπολέμηση αλλεργιών, για την καταστολή μετα- εμμηνοπαυσιακών συμπτωμάτων και καρδιαγγειακές θεραπείες. Επίσης, υπάρχει θετική συσχέτιση μεταξύ της πρόσληψης φλαβονοειδών και της μείωσης του κινδύνου για πρόκληση στεφανιαίας νόσου, εγκεφαλικών επεισοδίων και πολλών ειδών καρκίνου όπως έχει αποδειχθεί μέσα από πολυάριθμες επιδημιολογικές μελέτες. Πολλοί από τους δευτερογενείς μεταβολίτες που κατέχουν τις προαναφερθείσες ιδιότητες παράγονται από τα φυτά σε μικροποσότητες ή είναι δύσκολη η επεξεργασία και απομονωσή τους. Το ενδιαφέρον για την αξιοποίηση των ιδιοτήτων αυτών των δευτερογενών μεταβολιτών σε τομείς όπως η φαρμακοβιομηχανία, η ανθρώπινη υγεία, η γεωπονία (π.χ. βιολογική και ολοκληρωμένη φυτοπροστασία), έχει αυξήσει την ανάγκη για την ετερόλογη βιοσύνθεση των ουσιών που έχουν ή θεωρείται ότι έχουν επωφελείς ιδιότητες, όπως είναι τα φλαβονοειδή και τα στιλβενοειδή. Το κουμαρικό οξύ1, ένα φαινολικό οξύ, η ρεσβερατρόλη2, ένα στιλβενοειδές, η ναριγκενίνη3, μια φλαβανόνη, η γενιστεΐνη, μια ισοφλαβόνη και οι φλαβονόλες καμφερόλη και κερκετίνη, έχει αποδειχθεί ότι ανήκουν σε ουσίες με υψηλή διατροφική και αγρονομική αξία

Evolution of Physicochemical Properties and Phenolic Maturity of Vilana, Vidiano, Kotsifali and Mandilari Wine Grape Cultivars (<i>Vitis vinifera</i> 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

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

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

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