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.     

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.     

Differential ecophysiological responses to seasonal drought of three co-existing oak species in northern Greece

Oaks (Quercus spp.) represent the most important broadleaf genus with respect to forest-shaping tree species in the Mediterranean. Considering future climate scenarios (increased drought conditions), the identification of drought tolerant oak species is of great importance for future forest management in this region. The objective of the study was the comparison of physiological status of three economically and ecologically valuable oak species (Quercus ilex, Quercus frainetto and Quercus pubescens) co-existing in natural coppice stands in NE Greece, in response to seasonal drought stress. Measurements were conducted between June and September 2016, every 15–20 days until leaf falling. The parameters studied were predawn leaf water potential and fast chlorophyll fluorescence induction curves (OJIP test), chlorophyll content, and relative water content. Meteorological data from the area were also collected. Photosynthetic parameters such as performance indices (PI_abs and PI_tot) reacted to summer drought conditions, with Q. frainetto showing the lowest values. The discrepancy between species increased with duration of drought period. Q. frainetto revealed the lowest predawn water potential values. The results indicate that Q. frainetto is less suitable for future forestry applications in the studied climate/elevation zone than Q. pubescens and Q. ilex.     

Circumventing senescence is associated with stem cell properties and metformin sensitivity

Most cancers arise in old individuals, which also accumulate senescent cells. Cellular senescence can be experimentally induced by expression of oncogenes or telomere shortening during serial passage in culture. In vivo, precursor lesions of several cancer types accumulate senescent cells, which are thought to represent a barrier to malignant progression and a response to the aberrant activation of growth signaling pathways by oncogenes (oncogene toxicity). Here, we sought to define gene expression changes associated with cells that bypass senescence induced by oncogenic RAS. In the context of pancreatic ductal adenocarcinoma (PDAC), oncogenic KRAS induces benign pancreatic intraepithelial neoplasias (PanINs), which exhibit features of oncogene‐induced senescence. We found that the bypass of senescence in PanINs leads to malignant PDAC cells characterized by gene signatures of epithelial‐mesenchymal transition, stem cells, and mitochondria. Stem cell properties were similarly acquired in PanIN cells treated with LPS, and in primary fibroblasts and mammary epithelial cells that bypassed Ras‐induced senescence after reduction of ERK signaling. Intriguingly, maintenance of cells that circumvented senescence and acquired stem cell properties was blocked by metformin, an inhibitor of complex I of the electron transport chain or depletion of STAT3, a protein required for mitochondrial functions and stemness. Thus, our studies link bypass of senescence in premalignant lesions to loss of differentiation, acquisition of stemness features, and increased reliance on mitochondrial functions.     

Kinetic characterization of galacto‐oligosaccharide (GOS) synthesis by three commercially important β‐galactosidases

Many β‐galactosidases show large differences in galacto‐oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various β‐galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the β‐galactosidase of Bacillus circulans than the β‐galactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the β‐galactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why β‐galactosidase of B. circulans gives higher yields in GOS production than other β‐galactosidases. Many of the reaction rate constants for the β‐galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform β‐gal‐A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:38–47, 2014     

The C-terminal Region of HPNAP Activates Neutrophils and Promotes Their Adhesion to Endothelial Cells

Entire Helicobacter Pylori Neutrophil Activated Protein (HPNAP) and its truncated forms NH₂-terminal region HPNAP_1–57 and C-terminal region HPNAP_58–144 after cloning into pET29c vector, purification and removal of LPS traces were subjected to human neutrophil activation. Our results revealed that the C-terminal region of HPNAP is indispensable for human neutrophil stimulation and their further adhesion to endothelial cells – a step necessary to H. pylori inflammation – in a ratio equal to that exhibited by the entire protein.
In addition, experiments concerning the implication of Arabino-Galactan-Proteins (AGPs) derived from Chios Mastic Gum (CMG), the natural resin of the plant Pistacia lentiscus var. Chia revealed the inhibition of neutrophil activation and therefore their adhesion to endothelial cells, in vitro .
Both, the involvement of HPNAP C-terminal region in stimulation-adhesion of neutrophils to endothelial cells as well as the inhibition of this process by AGPs have to be further investigated and may be exploited in a future anti-inflammatory therapy for H. pylori patients.     

Use of Preclinical Dog Studies and Absorption Modeling to Facilitate Late Stage Formulation Bridging for a BCS II Drug Candidate

Formulation changes are common during drug development either due to clinical or manufacturing considerations. These changes especially at later stages of drug development oftentimes raise questions on the potential impact of a new formulation on bioavailability. In this work, the preclinical assessment of formulation bridging risk for a Biopharmaceutics Classification System II development compound is presented. Early clinical studies were conducted using a liquid-filled capsule (LFC). To assess the feasibility of a conventional solid dosage form, an initial analysis was conducted using absorption modeling which indicated conventional formulation of micronized active pharmaceutical ingredient (API) could be a viable option. Subsequently, test formulations were prepared and tested in vivo in dogs. The solid formulations were able to match exposures of the LFC capsule in the dog model; in addition, a sensitivity to API PSD was observed in line with the modeling predictions. When tested in the clinic, the conventional solid formulation resulted in exposures of approximately 25% lower compared to the LFC on an equivalent dose basis; however, bridging with a small dose adjustment would be feasible. The outcome of the clinical study was better predicted by the modeling approach while the dog model appeared to somewhat overestimate absorption. Through the use of preclinical tools and modeling and simulation, a risk assessment around formulation bridging can be conducted and inform formulation decisions or subsequent clinical study designs.     

Genetic relationship between Salix exigua and other North American willows (Salix L.): Evidence from allozyme variation

Starch gel electrophoresis was used to examine the allozyme variation and the genetic relationship between Salix exigua and other North American willows. A total of 149 clonal collections comprising six willow species were analyzed for 26 putative isozyme loci. Genetic distances between species were estimated and used for distance analysis of species relationships. Individual alleles were used as characters for cladistic analysis of phylogenetic inference. Distance analysis indicates that S. exigua has approximately equal divergence to both subgenus Salix and Vetrix. Results of cladistic analysis suggest that S. exigua may be a separate group isolated from all other willows. Based on evidence of allozyme variation, it is suggested that the taxonomic placement of S. exigua within subgenus Salix need to be revised.