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

LOPAT tests of eighteen local (Crete, Greece) <i>P. viridiflava</i> isolates along with <i>P. viridiflava</i> reference strain NCPPB1249 and other pseudomonads.

<p>LOPAT tests of eighteen local (Crete, Greece) <i>P. viridiflava</i> isolates along with <i>P. viridiflava</i> reference strain NCPPB1249 and other pseudomonads.</p

<i>P. viridiflava</i> phylogenetic trees, utilizing <i>rpoD</i> sequences along with sequences obtained from GenBank.

<p>The evolutionary history was inferred using the Neighbor-Joining method. Tree construction and evolutionary distances were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-g002" target="_blank">Figure 2</a> legend. The analysis involved 32 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 513 positions in the final dataset. The methodology used for the evolutionary analysis, tree construction and other details are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-g003" target="_blank">Figure 3</a> legend.</p

<i>P. viridiflava</i> phylogenetic trees, utilizing <i>rpoB</i> sequences along with sequences obtained from GenBank.

<p>The evolutionary history was inferred using the Neighbor-Joining method. Tree construction and evolutionary distances were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-g002" target="_blank">Figure 2</a> legend. The analysis involved 27 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 741 positions in the final dataset. The methodology used for the evolutionary analysis, tree construction and other details are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-g003" target="_blank">Figure 3</a> legend.</p

Analysis of Wine-Producing <em>Vitis vinifera</em> 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

<i>P. viridiflava</i> phylogenetic tree, utilizing <i>gyrB</i> sequences determined in this study along with sequences obtained from GenBank.

<p>The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1500 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 52 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 740 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. The host plant species is presented next to the code number (e.g. PVXXX) of each isolate.</p

Phylogenetic trees of the local <i>P. viridiflava</i> isolates.

<p>The construction of the dendrograms was based on <b>A</b>: BOX- and ERIC-PCR fingerprints (rep-PCR) and <b>B</b>: the combined <i>gyrB, rpoD</i> and <i>rpoB</i> gene sequences. The plant hosts are given next to the code number (PVXXX, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036090#pone-0036090-t001" target="_blank">Table 1</a>) of each isolate. The evolutionary history was inferred using the UPGMA method. The consensus tree inferred from 1500 replicates is taken to represent the evolutionary history of the isolates analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 2222 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.</p