Metabolic engineering in Nicotiana benthamiana reveals key enzyme functions in Arabidopsis indole glucosinolate modification

Indole glucosinolates, derived from the amino acid Trp, are plant secondary metabolites that mediate numerous biological interactions between cruciferous plants and their natural enemies, such as herbivorous insects, pathogens, and other pests. While the genes and enzymes involved in the Arabidopsis thaliana core biosynthetic pathway, leading to indol-3-yl-methyl glucosinolate (I3M), have been identified and characterized, the genes and gene products responsible for modification reactions of the indole ring are largely unknown. Here, we combine the analysis of Arabidopsis mutant lines with a bioengineering approach to clarify which genes are involved in the remaining biosynthetic steps in indole glucosinolate modification. We engineered the indole glucosinolate biosynthesis pathway into Nicotiana benthamiana, showing that it is possible to produce indole glucosinolates in a noncruciferous plant. Building upon this setup, we demonstrate that all members of a small gene subfamily of cytochrome P450 monooxygenases, CYP81Fs, are capable of carrying out hydroxylation reactions of the glucosinolate indole ring, leading from I3M to 4-hydroxy-indol-3-yl-methyl and/or 1-hydroxy-indol-3-yl-methyl glucosinolate intermediates, and that these hydroxy intermediates are converted to 4-methoxy-indol-3-yl-methyl and 1-methoxy-indol-3-yl-methyl glucosinolates by either of two family 2 O-methyltransferases, termed indole glucosinolate methyltransferase 1 (IGMT1) and IGMT2.     

EFFICIENT PRODUCTION OF Staphylococcus simulans LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT Escherichia coli

Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous, Escherichia coli expression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.     

Current state and prospects of biotechnology in Central and Eastern European countries. Part I: Visegrad countries (CZ,H, PL,SK)

Innovation is a key determinant of sustainable growth. Biotechnology (BT) is one such industry that has witnessed a revolution in innovative ideas leading to the founding of many new companies based on providing products, solutions and services, stretching from the food industry to environmental remediation, and new medicines. BT holds much promise for the development of national and local economies, however, this requires a strategic approach involving actors within government, industry, and academia working in concert to maximize this potential. This first article reviews the current “state of play” in the field of BT within the Central Eastern European (CEE) countries. For the purposes of this article, CEE refers to the countries of Czech Republic, Hungary, Poland, and Slovakia (the so-called Visegrad – V4 countries). We examine the components that support the creation and development of a BT sector in CEE and also highlight the barriers to these objectives. Clearly setting priorities for the countries’ policy agenda, as well as the alignment of Smart Specialization Strategy will help to focus efforts. Recent investments in R&D infrastructure within CEE have been substantial, but conditions will need to be optimized to harness these largely European investments for effective use towards SME high-tech development.     

Primate vocal communication: a useful tool for understanding human speech and language evolution?

Language is a uniquely human trait, and questions of how and why it evolved have been intriguing scientists for years. Nonhuman primates (primates) are our closest living relatives, and their behavior can be used to estimate the capacities of our extinct ancestors. As humans and many primate species rely on vocalizations as their primary mode of communication, the vocal behavior of primates has been an obvious target for studies investigating the evolutionary roots of human speech and language. By studying the similarities and differences between human and primate vocalizations, comparative research has the potential to clarify the evolutionary processes that shaped human speech and language. This review examines some of the seminal and recent studies that contribute to our knowledge regarding the link between primate calls and human language and speech. We focus on three main aspects of primate vocal behavior: functional reference, call combinations, and vocal learning. Studies in these areas indicate that despite important differences, primate vocal communication exhibits some key features characterizing human language. They also indicate, however, that some critical aspects of speech, such as vocal plasticity, are not shared with our primate cousins. We conclude that comparative research on primate vocal behavior is a very promising tool for deepening our understanding of the evolution of human speech and language, but much is still to be done as many aspects of monkey and ape vocalizations remain largely unexplored.     

Structure of the N-terminal domain of the FOP (FGFR1OP) protein and implications for its dimerization and centrosomal localization

The fibroblast growth factor receptor 1 (FGFR1) oncogene partner, FOP, is a centrosomal protein that is involved in the anchoring of microtubules (MTS) to subcellular structures. The protein was originally discovered as a fusion partner with FGFR1 in oncoproteins that give rise to stem cell myeloproliferative disorders. A subsequent proteomics screen identified FOP as a component of the centrosome. FOP contains a Lis-homology (LisH) motif found in more than 100 eukaryotic proteins. LisH motifs are believed to be involved in microtubule dynamics and organization, cell migration, and chromosome segregation; several of them are associated with genetic diseases. We report here a 1.6A resolution crystal structure of the N-terminal dimerization domain of FOP. The structure comprises an alpha-helical bundle composed of two antiparallel chains, each of them having five alpha-helices. The central part of the dimer contains the LisH domain. We further determined that the FOP LisH domain is part of a longer N-terminal segment that is required, albeit not sufficient, for dimerization and centrosomal localization of FOP.     

Protective effects of resveratrol against oxidative/nitrative modifications of plasma proteins and lipids exposed to peroxynitrite

The protective effects of resveratrol (3, 4', 5-trihydroxystilbene; present naturally in different plants) against the oxidative/nitrative damage of human plasma proteins induced by peroxynitrite (ONOO-) were studied and compared with those of deferoxamine (DFO; a natural siderophore isolated from Streptomyces pilosus), which is a typical and well-known antioxidant. We also studied the effect of ONOO- on plasma lipid peroxidation and the role of tested antioxidants in this process. ONOO- at the used concentrations (0.01-1 mM) showed toxicity to human plasma components. Exposure of plasma to ONOO- (0.1 mM) resulted in an increase of the level of carbonyl groups and nitrotyrosine residues in plasma proteins (approximately 4-fold and 76-fold, respectively) and in a distinct augmentation of lipid peroxidation (approximately 2-fold). In the presence of 0.1-mM resveratrol, a distinct decrease of carbonyl group formation and tyrosine nitration in plasma proteins caused by 0.1-mM ONOO- was observed (by approximately 70% and 65%, respectively). Addition of 0.1-mM DFO to plasma also distinctly reduced the level of carbonyl groups and nitrotyrosines caused by 0.1-mM ONOO- (by approximately 50% and 60%, respectively). Moreover, these antioxidants also inhibited plasma lipid peroxidation induced by ONOO- (0.1 mM). The obtained results indicate that in vitro resveratrol, like well-known antioxidant DFO, has inhibitory effects on ONOO- -mediated oxidation of proteins and lipids in human plasma.     

Effects of boron fertilization on `Conference' pear tree vigor, nutrition, and fruit yield and storability

The aim of the study was to examine the response of pear (Pyrus communis L.) trees to soil and foliar applications of boron (B). The experiment was carried out during 2000–2001 in a commercial orchard in Central Poland on mature `Conference' pear trees grafted on Pyrus communis var. caucasica seedlings planted at a spacing of 4 × 2.5 m on a sandy loam soil with a low hot water-extractable B status. Annually, foliar sprays with B were applied. (i) before full bloom (at green and white bud stage, and when 1–5% of flowers was at full bloom), (ii) after flowering (at petal fall, and 7 and 14 days after the end of flowering), or (iii) postharvest in fall (approximately 6 weeks before leaf fall). Spray treatments involved application of B at a rate of 0.2 kg ha^–1 in spring or 0.8 kg ha^–1 in fall. Additionally, other trees were supplied with soil-applied B at the bud break stage at a rate of 2 kg ha^–1. Trees untreated with B served as the control. The results revealed that foliar applications of B before full bloom or after harvest increased fruit set and fruit yield. Tree vigor, mean fruit weight, firmness, soluble solids concentration and titratable acidity of fruits at harvest were not affected by B treatments. Foliar B sprays before full bloom or after harvest increased B concentrations in flowers, and both leaves and fruitlets at 40 days after flowering. Only the foliar treatments after flowering and soil fertilization with B increased the content of this microelement in fruit and leaves at 80 and 120 days after full bloom. Foliar B application before full bloom or after harvest increased calcium (Ca) in fruitlets at 40 days after full bloom, in fruit, and in leaves at 80 and 120 days after full bloom. Nitrogen (N), phosphorus (P), potassium (K), and magnesium (Mg) in plant tissues were not affected by B fertilization. After storage, and also after the ripening period, fruits from the trees sprayed with B before full bloom or after harvest had higher firmness and titratable acidity than those from the control trees. After the ripening period, fruits from the trees sprayed with B before full bloom or after harvest had lower membrane permeability and were less sensitive to internal browning than the control fruits. These findings indicate that prebloom and postharvest B sprays are successful in increasing pear tree yielding and in improving fruit storability under the conditions of low B availability in the soil.     

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect

A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25-30% (130-150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.