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.

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.

Analysis of Wine-Producing Vitis vinifera L. Biotypes, Autochthonous to Crete (Greece), Employing Ampelographic and Microsatellite Markers.

Vitis vinifera ssp. vinifera (domesticated grapevine) includes thousands of cultivars, which are classified according to their main uses, as wines, fresh fruits or dried raisins and sultanas since ancient times. Evidence showed that Crete grapevine cultivars and winemaking date back to 2300 BC. In this study, fifty-one genotypes belonging to seven different traditional Vitis vinifera cultivars, presumed autochthonous to the island of Crete, were selected for their wine-producing potential and classified by 51 ampelographic descriptors. In addition, five genotypes belonging to two non-autochthonous cultivars were included as out-group controls. Subsequently, in order to characterize genetic diversity, establish genetic relationships within and between cultivars and solve accession-labeling problems, genotypes were fingerprinted employing Simple Sequence Repeat (SSR or microsatellite) markers. Four of the autochthonous cultivars namely 'Vidiano', 'Vilana', 'Plyto', and 'Moschato Spinas' are used in the local economy for blanc (white) wine production while the rest, namely 'Kotsifali', 'Liatiko' and 'Mantilari' for Noir (red) wines. The two cultivars employed as out-group were 'Moschato Samou' and 'Moschato Alexandrias': both white wine producers. Ampelography-based clustering grouped the majority of genotypes along cultivar-specific clusters. All three Moschato cultivars formed a distinct clade pointing to the non-autochthonous origin of 'Moschato Spinas'. A total of one hundred and thirteen (113) SSR alleles were amplified from thirteen (13) SSR loci, with an average number of alleles per locus equal to 10.23 revealing ample genetic polymorphism. The cumulative probability of identity was also quite high (3.389 × 10-16). The overall observed heterozygosity was 0.837 while for twenty-nine of the examined genotypes, at least one private SSR allele was detected. The majority of genotypes were grouped in cultivar-specific clusters. The results of this paper pave the way for the certification and registration of clones of some of the most important wine-producing cultivars in Crete.

The LuxR Regulators PcoR and RfiA Co-regulate Antimicrobial Peptide and Alginate Production in Pseudomonas corrugata.

Cyclic lipopeptides (CLPs) are considered as some of the most important secondary metabolites in different plant-associated bacteria, thanks to their antimicrobial, cytotoxic, and surfactant properties. In this study, our aim was to investigate the role of the Quorum Sensing (QS) system, PcoI/PcoR, and the LuxR-type transcriptional regulator RfiA in CLP production in the phytopatogenic bacterium, Pseudomonas corrugata based on our previous work where we reported that the pcoR and rfiA mutants were devoid of the CLPs cormycin and corpeptin production. Due to the close genetic link between the QS system and the RfiA (rfiA is co-transcribed with pcoI), it was difficult to ascertain the specific regulatory role in the expression of target genes. A transcriptional approach was undertaken to identify the specific role of the PcoR and RfiA transcriptional regulators for the expression of genes involved in CLP production. The RNA-seq-based transcriptional analysis of the wild-type (WT) strain CFBP 5454 in comparison with GL2 (pcoR mutant) and GLRFIA (rfiA mutant) was performed in cultural conditions favoring CLP production. Differential gene expression revealed that 152 and 130 genes have significantly different levels of expression in the pcoR and rfiA mutants, respectively. Of these, the genes linked to the biosynthesis of CLPs and alginate were positively controlled by both PcoR and RfiA. Blast homology analysis showed that 19 genes in a large CLP biosynthetic cluster involved in the production of three antimicrobial peptides, which span approximately 3.5% of the genome, are strongly over-expressed in the WT strain. Thus, PcoR and RfiA function mainly as activators in the production of bioactive CLPs, in agreement with phenotype analysis of mutants. RNA-seq also revealed that almost all the genes in the structural/biosynthetic cluster of alginate exopolysaccharide (EPS) are under the control of the PcoR-RfiA regulon, as supported by the 10-fold reduction in total EPS yield isolated in both mutants in comparison to the parent strain. A total of 68 and 38 gene expressions was independently regulated by PcoR or RfiA proteins, respectively, but at low level. qPCR experiments suggest that growth medium and plant environment influence the expression of CLP and alginate genes.

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.