Day 1 :
Keynote Forum
Peter J F Henderson
University of Leeds, UK
Keynote: Kinetic and molecular dissection of coupled ion-substrate membrane transport proteins
Time : 10:00-10:40
Biography:
Peter J F Henderson is a Professor of Biochemistry and Molecular Biology in the University of Leeds. He obtained his BSc in 1965 and PhD in 1968, both in Biochemistry, at the University of Bristol. After Postdoctoral training at the Enzyme Institute, Madison, University of Wisconsin and in the Department of Biochemistry at Leicester, he became a University Lecturer in 1973. In 1975 he moved to the Department of Biochemistry at Cambridge, where he became Reader in Molecular Biology of Membranes in 1990. He has held Visiting Professorships in Japan, Canada and Australia. He was Scientific Director of the European Membrane Protein (EMeP) consortium 2003-2008, Coordinator of the European Drug Initiative for Channels and Transporters (EDICT) 2008-2012 and held Leverhulme Trust Emeritus Research Fellowships in 2001-2002 and 2014-2017. He has published over 200 scientific papers in the fields of Membrane Transport, Enzyme Kinetics and Structural Biology.
Abstract:
The Mhp1 Na+-hydantoin membrane symport protein from Microbacterium liquefaciens is a paradigm for the nucleobase-cation-symport, NCS-1, family of transport proteins found widely in archaebacteria, bacteria, yeasts and plants. Their metabolic roles include the capture by cells of nitrogen compounds and vitamins from the environment. Mhp1 is also a structural model for the huge range of ‘5-helix-inverted-repeat’ superfamily of proteins, because, unusually, crystal structures are available for its open-outwards, occluded, and open-inward conformations. Here we accomplish a detailed dynamic model of the partial reactions in an alternating access cycle of membrane transport derived from substrate binding studies to the purified Mhp1 protein by combining novel mass spectrometry, stopped-flow and steady state kinetic analyses and mutagenesis. The mechanism of coupling substrate transport to the Na+-gradient is revealed during a sequence of mostly reversible kinetic steps that explain how transfer of substrate across the membrane is affected by changes in conformational states. The AceI H+/substrate antiport protein from Acinetobacter baumannii is a paradigm for the proteobacterial antimicrobial compound efflux (PACE) family of drug efflux proteins found dispersed throughout the Proteobacteria. AceI contributes to the resistance of Acinetobacter baumannii towards the widely used antiseptic, chlorhexidine. Currently there is little structural information about the PACE family of transport proteins, but progress towards understanding the recognition of substrates and cations by AceI and its homologues will be discussed.
Keynote Forum
Magali Remaud Simeon
INSA - Université de Toulouse, France
Keynote: Mixing enzyme discovery with engineering for sucrose-derived bioproducts: the case of GH13 and GH70 polymerases
Time : 10:40-11:20
Biography:
Magali Remaud Simeon is Professor at the National Institute of Applied Sciences of Toulouse and is head of the Catalysis and Enzyme Molecular Engineering group of the “Laboratoire d’Ingénierie des Système Biologiques and Procédé (LISBP). She received her PhD in Biochemistry from the University of Toulouse and was Post-Doc at the University of Pennsylvania. She has co-authored more than 150 papers and is co-inventor of 22 patents. Her research activities focus on Enzyme Engineering for white biotechnology, green chemistry, health, food/feed industries and synthetic biology. They cover enzyme structure/activity relationship studies, kinetic resolution, evolution combining both rational and combinatorial approaches, and applications to the synthesis of glycans, glycoconjugates and various synthons of interest. Her work is currently focused on the search and generation of enzymes displaying new specificities and improved catalytic properties. Her objective is to open new trajectories for biomass transformation. To this end, she specifically targets the integration of tailored enzymes in chemo-enzymatic cascades, new metabolic pathways or enzyme-based processes.
Abstract:
The exploration of the natural diversity, through data mining, functional genomics and/or metagenomics is an efficient mean to discover enzymes showing new functions or improved performances. These approaches can be further completed or run in parallel with semi-rational protein engineering based on structure/function studies or directed molecular evolution inspired from nature. Which of these alternatives are the best ones, in terms of effort, rapidity and efficiency? This is an open question to which a definite answer can be hardly formulated a priori. For illustration, we will take a few examples from our most recent work on glucansucrases from GH13 and GH70 families. These enzymes are naturally very efficient transglucosylases. They use sucrose as substrate and catalyze polymerization of its glucosyl units as a main reaction. Depending on their specificity, structures varying in size as well as in glycosidic linkage types can be obtained, thus giving access to an interesting panel of biopolymers. A campaign of genome sequencing and data mining allowed the isolation of atypical enzymes with new product specificities. In particular, a hyper efficient polymerase producing a gel-like polymer and, in contrast an enzyme synthesizing directly from sucrose a polymer of well-controlled low molar mass could be characterized. Structure-function studies combined with mutagenesis assays allowed us to decipher some of the molecular mechanisms behind the control of the polymer size and enzyme processivity. Another key property of these catalysts is coming from their ability to glucosylate a broad spectrum of hydroxylated molecules. Computational protein design, structurally-guided engineering and also random approaches such as neutral evolution was implemented for a fine tuning of their acceptor specificity toward non-natural acceptors such chemically protected disaccharides for vaccinal applications, polyol, flavonoids, or various chemicals. These various approaches will be described and discussed with regard to the engineering objectives.
Keynote Forum
Sergey Suchkov
I M Sechenov First Moscow State Medical University, Russia
Keynote: Proteolytic abzymes as translational tools of the newest generation to be exploited for bi-odesign and bioengineering
Time : 11:10-11:50
Biography:
Catalytic Abs (catAbs) are multivalent immunoglobulins (Igs) with a capacity to hydrolyze the antigenic (Ag) substrate. In this sense, proteolytic Abs (Ab-proteases) represents Abs to provide proteolytic effects. Abs against myelin basic protein/MBP with proteolytic activity exhibiting sequence-specific cleavage of MBP is of great value to monitor demyelination whilst in multiple sclerosis. The activity of Ab-proteases was first registered at the subclinical stages, 1-2 years prior to the clinical illness and the activity of the Ab-proteases revealed significant correlation with scales of demyelination and the disability of the patients as well. So, the activity of Ab-proteases and its dynamics tested would confirm a high subclinical and predictive (translational) value of the tools as applicable for personalized monitoring protocols. Ab-proteases directly affecting remodeling of tissues with multilevel architectonics (for instance, myelin) are of tremendous value. By changing sequence specificity one may reach reduction of a density of the negative proteolytic effects within the myelin sheath and thus minimizing scales of demyelination. Ab-proteases can be programmed and re-programmed to suit the needs of the body metabolism or could be designed for the development of new catalysts with no natural counterparts. Further studies are needed to secure artificial or edited Ab-proteases as translational tools of the newest generation to diagnose, to monitor, to control and to treat and rehabilitate multiple sclerosis patients at clinical stages and to prevent the disorder at subclinical stages in persons at risks.
Abstract:
Catalytic Abs (catAbs) are multivalent immunoglobulins (Igs) with a capacity to hydrolyze the antigenic (Ag) substrate. In this sense, proteolytic Abs (Ab-proteases) represents Abs to provide proteolytic effects. Abs against myelin basic protein/MBP with proteolytic activity exhibiting sequence-specific cleavage of MBP is of great value to monitor demyelination whilst in multiple sclerosis. The activity of Ab-proteases was first registered at the subclinical stages, 1-2 years prior to the clinical illness and the activity of the Ab-proteases revealed significant correlation with scales of demyelination and the disability of the patients as well. So, the activity of Ab-proteases and its dynamics tested would confirm a high subclinical and predictive (translational) value of the tools as applicable for personalized monitoring protocols. Ab-proteases directly affecting remodeling of tissues with multilevel architectonics (for instance, myelin) are of tremendous value. By changing sequence specificity one may reach reduction of a density of the negative proteolytic effects within the myelin sheath and thus minimizing scales of demyelination. Ab-proteases can be programmed and re-programmed to suit the needs of the body metabolism or could be designed for the development of new catalysts with no natural counterparts. Further studies are needed to secure artificial or edited Ab-proteases as translational tools of the newest generation to diagnose, to monitor, to control and to treat and rehabilitate multiple sclerosis patients at clinical stages and to prevent the disorder at subclinical stages in persons at risks.
References:
1. Gabibov A A, Paltsev M A and Suchkov S V (2011) Antibody-associated proteolysis in surveillance of autoimmune demyelination: clinical and preclinical issues. Future Neurology 6(3):303-305.
2. D Kostyushev, I Tsarev, D Gnatenko, M Paltsev and S Suchkov (2011) Myelin-associated serological targets as applicable to diagnostic tools to be used at the preclinical and transient stages of multiple sclerosis progression. Open J Immunology 1(3):80-86.
3. Gabibov A G, Ponomarenko N A, Tretyak E B, Paltsev M A and Suchkov S V (2006) Catalytic autoantibodies in clinical autoimmunity and modern medicine. Autoimmunity Reviews 2006(5):324-330.
4. Ponomarenko N A, Durova O M, Vorobiev I I, Belogurov A A, Telegin G B, et al. (2005) Catalytic activity of autoantibodies toward myelin basic protein correlates with the scores on the multiple sclerosis expanded disability status scale. Immunol. Lett. 103(1):45-50.
5. Ponomarenko N A, Durova O M, Vorobiev I I, Aleksandrova E S, Telegin G B, et al. (2002) Catalytic antibodies in clinical and experimental pathology: human and mouse models. Journal of Immunological Methods 2002(269):197-211.
Keynote Forum
David Rabuka
Catalent Biologics, USA
Keynote: Developing site-specifically modified ADCs using a chemoenzymatic approach
Time : 11:50-12:30
Biography:
David Rabuka received a PhD in Chemistry at the University of California, Berkeley as a Chevron Fellow in the Lab of Carolyn Bertozzi. His research included developing and applying the SMARTagTM platform technology to cell surface modification. Prior to joining Bertozzi’s lab, he worked at the Burnham Institute synthesizing complex glycans followed by Optimer Pharmaceuticals, where he focused on the development of glycan and macrolide based antibiotics. He was CSO, President and Co-founder of Redwood Bioscience, where he developed novel protein conjugation methods and biotherapeutic applications such as antibody-drug conjugates. Redwood Bioscience was acquired by Catalent Pharma Solutions in Oct 2014, where he has continued to apply the SMARTagTM technology with various collaborators and partners as a Global Head of R&D. He graduated with a Double Honors BS in Chemistry and Biochemistry from the University of Saskatchewan, where he received the Dean’s Science Award, and holds an MS in Chemistry From the University of Alberta. He has authored over 45 major publications, as well as numerous book chapters and holds over 30 patents.
Abstract:
We have developed the SMARTagTM technology platform, which enables precise, programmable, site-selective chemical protein modification. Leveraging the target sequence of formylglycine generating enzyme (FGE), we chemoenzymatically modify proteins to generate a precisely placed aldehyde functionality that can be chemically elaborated. Subsequently, novel ligation chemistry is employed that exploits this “aldehyde tag” site. We will present recent data on our novel protein modification platform and its application to generating novel bioconjugates, including ADCs, utilizing our new conjugation chemistries and linkers. The application of these chemistries to generate site-specifically modified bioconjugates with improved efficacy and safety profiles will be presented. Additionally, we will highlight the progress in developing conjugates with a focus on preclinical studies as well as highlight our progress in cell line development and manufacturing by using this chemoenzymatic approach.
- Structural Enzymology | Enzymology & Biochemistry
Location: Armstrong
Session Introduction
Albert Jeltsch
University Stuttgart, Germany
Title: Molecular enzymology of DNA methyltransferases – conformational changes and allosteric regulation
Time : 15:30-16:00
Biography:
Albert Jeltsch completed his PhD working on the mechanism of restriction endonucleases at University of Hannover in 1994. Afterwards, he started to study DNA methyltransferases at Justus-Liebig University Giessen and at Jacobs University Bremen. Since 2011, he is a Professor of Biochemistry at the University Stuttgart. He received the Gerhard-Hess award (DFG) and BioFuture award (BMBF). He has long standing expertise in Biochemical study of DNA and protein methyltransferases, methyl lysine reading domains and in rational and evolutionary protein design. His work has been published in more than 250 publications in peer reviewed journals and he is in the editorial boards of several journals.
Abstract:
DNA methylation is an essential epigenetic chromatin modification. The setup and maintenance of DNA methylation patterns depends on the coordinated activity of DNA methyltransferases (DNMTs) and their allosteric regulation by interacting proteins, other chromatin modifications and post-translational modifications. I will present novel assays for DNMTs including single enzyme assays to study their mechanism and conformationally locked mutants to study allosteric effects. Based on this, recent data regarding the regulation and targeting of DNMTs by allosteric effect will be presented. Moreover, I will present insights into the mechanism of DNMTs regarding target site location, specificity and processivity.
David J Merkler
University of South Florida, USA
Title: N-acyltransferases and their role in fatty acid amide biosynthesis
Time : 16:20-16:50
Biography:
David J Merkler obtained a PhD in Biochemistry from Pennsylvania State University in 1985 and completed Postdoctoral Fellowships in Enzymology at Temple University School of Medicine (1985-1987) and the Albert Einstein College of Medicine (1987-1989). His next position was as Senior Scientist at Unigene Laboratories, Inc. involved in the in vitro production of a peptide hormone, calcitonin. In 1995, he moved back to academia as a Professor of Chemistry and Biochemistry first at Duquesne University (1995-1999) and then the University of South Florida (1999-present). His laboratory has been interested in the fatty amides: identification and characterization of the fatty acid amides (Lipidomics), identification and characterization of the enzymes of fatty acid amide biosynthesis (Enzymology and Structural Biology), and changes in the fatty acid amidome after targeted enzyme knock-out (subtraction lipidomics).
Abstract:
Fatty acid amides are a family of cell signaling lipids with the general structure of R-CO-NH-Y. This structural simplicity belies a wealth of diversity amongst this lipid family as the R-group is derived from fatty acids (R-COOH) and the Y-group is derived from biogenic amines(H2N-Y). The fatty acid amide family is divided into classes, defined by parent amines. Examples include the N-acylethanolamines (NAEs, R-CO-NH-CH2-CH2OH) and the N-acylglycines (NAGs,R-CO-NH-CH2-COOH). Other classes of fatty acid amides are known. The best known fatty acid amide is N-arachidonoylethanolamine (anandamide), a fatty acid amide found in the human brain that binds to the cannabinoid receptors. We have a long interest in the enzymes of fatty acid amide biosynthesis. We identified an enzyme that oxidizes the NAGs to the primary fatty acid amides and showed that inhibiting this enzyme led to the cellular accumulation of the NAGs. We have characterized several insect N-acyltransferases (from D. melanogaster, B. mori, and T. castaneum) that catalyze the acyl-CoA-dependent formation of fatty acid amides from an amine acyl-acceptor substrate. Knock-out experiments in D. melanogaster validate our in vitro substrate specific studies demonstrating that one novel N-acyltransferases, arylalkylN-acyltransferase-like 2 (AANATL2), does catalyze the formation ofN-acyldopamines in vivo. We developed a straightforward platform technology to rapidly identify substrates for our panel of uncharacterized insect N-acyltransferases. Our application of this technology leads to identification of an enzyme in D. melanogaster, agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine, a virtually unknown metabolite. We have determined the X-ray structure of AgmNAT. Our work on AgmNAT hints at an unknown reaction in arginine metabolism and points to a novel class on fatty acid amides, the N-acylagmatine. The presentation will also include our results on the kinetic and chemical mechanisms of the novel N-acyltransferases.
Recent Publications
1. Dempsey D R et al. (2017) Structural and mechanistic analysis of Drosophila melanogaster agmatine N-acetyltransferase, an enzyme that catalyzes the formation of N-acetylagmatine. Sci. Rep. 7(1):13432.
2. Aboalroub A A et al. (2017) Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase. PLoS One. 12(5):e0177270.
3. Jeffries K A et al. (2016) Glycine N-acyltransferase-like 3 is responsible for long-chain N-acylglycine formation in N18TG2 cells. J. Lip. Res. 57(5):781-790.
4. Dempsey D R, Carpenter A M, Rodriguez Ospina S and Merkler D J (2015) Probing the chemical mechanism and critical regulatory amino acid residues in of Drosophila melanogaster arylalkylamine N-acyltransferase like 2. Insect Biochem. Mol. Biol. 66:1-12.
5. Dempsey D R et al. (2015) Mechanistic and structural analysis of a Drosophila melanogaster enzyme, arylalkylamine N-acetyltransferase like 7, an enzyme that catalyzes the formation of
N-acetylarylalkylamides and N-acetylhistamine. Biochemistry. 54(16):2644-2658.
Karlo M Lopez
California State University-Bakersfield, USA
Title: Lysyl oxidase: a versatile and elusive enzyme
Time : 16:50-17:20
Biography:
Karlo M Lopez is currently an Associate Professor of Biochemistry at California State University, Bakersfield. He received a PhD from Clark University and was a Howard Medical Institute Fellow at Pomona College. His research focuses primarily on the structural characterization of lysyl oxidase and understanding the role this enzyme plays in cancer metastasis. He is a member of the Committee on Ethics of the American Chemical Society and was part of the Task Force for Safety Education Guidelines.
Abstract:
Lysyl oxidase is an extracellular matrix, copper-dependent, amine oxidase that catalyzes a key crosslinking step in collagen and elastin. The enzyme is synthesized as a proenzyme that, upon excretion to the extracellular matrix, is cleaved at the Gly168-Asp169 bond by procollagen C-proteinase in the mammalian form of the enzyme. Lysyl oxidase is highly regulated and changes in its regulation have been shown to play a role in fibrosis and several other diseases. More recently, the enzyme has been shown to play a paradoxical role in cancer. In the early stages of cancer, the cleaved pro-peptide has been shown to inhibit the RAS oncogene, whereas in late stages of cancer lysyl oxidase has been shown to promote metastasis. Lysyl oxidase is highly insoluble and this has hampered its full characterization. Recent work in the by our study group has addressed some of the issues associated with the insolubility and characterization of the enzyme. In particular, this talk will address how plasmids were used to increase enzyme yields over those obtained directly from bovine aortic tissue, the role solubility tags play on enzyme activity and suitability for characterization studies, and will end with an innovative new approach to drug delivery that targets lysyl oxidase in cancer cells but remains inactive in normal cells.
References:
1. Oldfield R, Johnston K, Limones J, Ghilarducci C and Lopez K (2017) Identification of histidine 303 as the catalytic base of lysyl oxidase via site – directed mutagenesis. The Protein Journal, doi: 10.1007/s10930-017-9749-3.
2. Smith M A, Gonzalez J, Hussain A, Oldfield R N, Johnston K A, et al. (2016) Overexpression of soluble recombinant human lysyl oxidase by using solubility tags: effects on activity and solubility. Enzyme Research 2016:1-7.
3. Lopez K and Greenaway F T (2011) Identification of the copper-binding ligands of lysyl oxidase. Journal of Neural Transmission 118:1101-1109.
4. Herwald S, Greenaway F and Lopez K (2010) Purification of high yields of catalytically active lysyl oxidase directly from Escherichia coli cell culture. Protein Expression and Purification 74:116-121.
Anna V Hine
Aston University, UK
Title: ProxiMAX randomization: precision protein engineering
Time : 17:20-17:50
Biography:
Anna V Hine studied at the University of Manchester (UK) and Harvard Medical School. She is a Reader and Associate Dean Enterprise at Aston University (UK). In March 2013, she was named BBSRC Commercial Innovator of the Year 2013, for her work in transferring ProxiMAX randomization into SME Isogenica Ltd. She is a Molecular Biologist by training, she relishes interdisciplinary work.
Abstract:
ProxiMAX randomization is the technology that lies behind Isogenica’s Colibra™ offering. It is a defined saturation mutagenesis process that delivers precision control of both identity and relative ratio of amino acids at specified locations within a protein/antibody library. Thus unwanted amino acids such as cysteine and methionine can be eliminated from libraries because no constraints are imposed by the genetic code. Moreover, the process is non-degenerate, which means that encoding DNA libraries are as small as is physically possible. ProxiMAX relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle and is the science behind the commercial Colibra™ technology. Currently focused on antibody libraries but with achieved diversities of >99% (6 & 11 saturated codons) and the potential to generate libraries of up to 1014 components, we contest that ProxiMAX randomization is a vital tool in engineering any protein library of the highest quality. This presentation will examine the development of the ProxiMAX process and give examples of libraries created to date.
Reference:
1. Ferreira Amaral M M, Frigotto L and Hine A V (2017) Beyond the natural proteome: nondegenerate saturation mutagenesis - methodologies and advantages. Meth. Enyzmol. 585:111-133.
2. Frigotto L, Smith M E, Brankin C, Sedani A, Cooper S E, Kanwar N, Evans D, Svobodova S, Baar C, Glanville J, Ullman C G and Hine A V (2015) Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing. Antibodies 4:88-102.
3. Chimonides G F, Behrendt J M, Chundoo E, Bland C, Hine A V, Devitt A, Nagel D A and Sutherland A J (2014) Cellular uptake of ribonuclease A functionalised core–shell silica microspheres. J Mater Chem B, 2:7307-7315.
4. Nagel D, Behrendt J M, Chimonides G F, Torr E E, Devitt A, Sutherland A J and Hine A V (2014) Polymeric microspheres as protein transduction reagents. Mol. Cell Proteomics, 13:1543-1551.
5. Ashraf M, Frigotto L, Smith M E, Patel S, Hughes M D, Poole A J, Hebaishi H R M, Ullman C G and Hine A V (2013) ProxiMAX randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons. Biochem. Soc. Trans. 41:1189-1194.
Sotaro Fujii
Hiroshima University, Japan
Title: Stability and function of a thermophilic cytochrome c'
Biography:
Sotaro Fujii is now working on the stability, structure, and function of proteins that are important for microbial energy metabolism. A characteristic aspect of his research activity is comparison of the homologous proteins isolated from microorganisms living in extreme environments in which humans cannot live and those isolated from ‘normal’ environments.
Abstract:
Cytochromes c' are classified as heme proteins found in restricted Gram-negative bacteria. They usually form a homo dimeric structure, and the single subunit typically consists of four helix bundle. Biochemical analysis showed that they can bind diatomic gasses such as NO or CO, but not O2. Recently we purified cytochrome c' from thermophilic Hydrogenophilus thermoluteolus, and named it PHCP. H. thermoluteolus grows optimally at 52°C, indicating that PHCP is more stable than homologous proteins from mesophiles. In this study, we compared stability and function of PHCP with its mesophilic homologue, Allochromatium vinosum cytochrome c' (AVCP) having 55 % amino acid sequence identity. In order to check the stability, we measured the circular dichroism spectra with increasing temperature. The denaturation temperature of PHCP was 87°C, which was higher than that of AVCP (52°C). The X-ray structure comparison between PHCP and AVCP revealed that the stability difference was due to the heme-related interactions and subunit-subunit interactions, which was also proofed by mutagenesis study. These results indicated that PHCP advantageously retains the native structure at high temperature. The PHCP X-ray structure further revealed a ligand binding channel and a penta-coordinated heme, as observed in the AVCP protein, indicating PHCP could bind diatomic gasses at high temperature. Thus, we measured the gas binding affinity of PHCP and AVCP using absorption spectra. The association constant (Ka) of PHCP with CO was 3 times lower than that of AVCP at 25°C, and PHCP could maintain normal spectral changes up to 60°C. In AVCP, such spectral changes with CO could not to be detected at 60°C, because of denaturation of AVCP. In conclusion, PHCP has a structure fulfilling the requirement for both gas-binding function and thermal stability. This stable cytochrome c' will become a model for protein engineering field.
Iwona Żur
Polish Academy of Sciences, Poland
Title: Efficiency of the antioxidative system is the first prerequisite for effective doubled haploids production with the use of isolated microspore culture method
Biography:
Iwona Å»ur completed her PhD and Habilitation in the field of Agronomy and Plant Physiology at the University of Agriculture in Kraków, Poland. Since 2010, she has been the Head of the Department of Cell Biology at the Institute of Plant Physiology Polish Academy of Sciences. She has published 38 papers in peer-reviewed journals.
Abstract:
The technology of doubled haploids as the fastest route to total homozygosity is highly appreciated in many domains of basic research and breeding. Among several methods, the one using isolated and in vitro-cultured immature cells of male gametophyte induced towards embryogenic development (microspore embryogenesis-ME) possesses the highest potential for commercial application. However, efficient ME induction requires a precisely balanced stress treatment, strong enough to induce microspore reprogramming but not exceeding cell stress tolerance threshold. As the general cause of injuries in in vitro-cultured cells is the overproduction of reactive oxygen species (ROS), an efficient antioxidative defence was suggested as the first prerequisite for stress survival and effective ME initiation. To establish the role of ROS and the antioxidative system in ME initiation, the generation of hydrogen peroxide, and the activities of antioxidative enzymes and low molecular weight antioxidants were analysed in isolated microspores of two cultivars of barley (Hordeum vulgare L.), winter cv. Igri and spring cv. Golden Promise, differing significantly with respect to embryogenic potential. The analyses were conducted in microspores redirected towards embryogenic development by low temperature tillers pre-treatment (4 weeks at 4°C). Additionally, the effects of compounds known as cellular redox status modifiers, e.g. glutathione and L-2-oxo-4-thiazolidinecarboxylic acid (OTC), on microspore viability and ME initiation efficiency were estimated. The received results suggest that the activity of the antioxidative system is the first prerequisite for successful ME initiation, though in the case of its low activity, antioxidative defence could be supported by the application of exogenous antioxidants.
Omirbekova N Zh
Al-Farabi Kazakh National University, Kazakhstan
Title: Development of approach to obtain Brachypodium distachyon L. regenerative plants with morphogenetic stability
Biography:
Omirbekova N Zh graduated from Al-Farabi Kazakh National University and Lomonosov Moscow State University and has completed her Doctoral studies from Al-Farabi Kazakh National University. She is currently a Professor at the Department of Molecular Biology and Genetics, School of Biology and Biotechnology of KazNU named after Al-Farabi (Republic of Kazakhstan). Her research interests include chemical mutagenesis, genetics and biochemistry of wheat. She has published more than 30 papers in high valued journals.
Abstract:
The aim of the research is development of effective methodological approaches of in vitro cultivation, object 21 line (BD21) B. distachyon. In order to develop cultivation methods, ability for callus formation, regeneration of generative and vegetative organs of VD21 was studied. To cultivate, Linsmayer-Skoog and Murashige-Skoog medium, additional introduction of phytohormones was used. Aseptic culture conditions for callusogenesis cultivation: under dark conditions at a temperature of 24°C, for t shoots regeneration: 16/8 hour photoperiod and lighting of 3000 lux. Inflorescence and immature embryos isolated from green spikes of vegetating plants and isolated embryos from mature seeds were used as primary explants to induce callus formation in vitro. During immature embryo cultivation, callus formation takes place near the corimbe for 20-25 days. During the cultivation of whole caryopsis with mature embryos, the sprouts grew after a week of cultivation on MS medium without hormones. The level of maturity of isolated caryopsis has a significant influence on the callus formation and the type of callus tissue. The mature caryopsis formed callus on the 10th day of cultivation with a frequency of 75%. The cultivation of the overgrown caryopsis in the dark on medium MS 1 with 2 mg/L 2.4 DPA, led to the formation of a primary shoot in 60% of explants; the formation of callus in the area of the scute, but for 30-35 days. Passage of the callus on the same medium and on the hormone-free medium led to the appearance of greenish pointwise impregnation of 30% of the calluses. For microclonal propagation, nodal segments of young shoots of plants were introduced into the culture. To culture introduction, side shoots 5 cm long with 3-4 interstitial sites were cut, the microcrops were planted in inducing media. The shoot-forming capacity of primary explants was about 59%; the multiplication factor for two passages was 5.7.
References:
1. Omirbekova N, Kenzhebayeva S, Capstaff N, Fatma Sarsu, et al. (2017) Searching a spring wheat mutation resource for
correlations between yield, grain size, and quality parameters. Journal of Crop Improvement 31:209-228.
2. Omirbekova N, Kenzhebayeva S, Doktyrbay G et al. (2016) Frequency of vernalization requirement associated dominant
VRN-A1 gene and earliness related Esp-A1 candidate genes in advanced wheat mutant lines and effect of allele on flowering
time. International Journal of Biology and Chemistry 9:24-30.
3. Omirbekova N, Zhussupova A and Zhunusbayeva Zh (2015) Brachypodium distachyon as a model plant in wheat rust
research. International Journal of Biology and Chemistry 2:52-55.
Biography:
Helmi Mohamed El-bendary is Assistant Professor of Agriculture at Fayoum University. He finished his BSc in Plant Protection Department at Cairo University and DSPU at Mediterranean Agronomic Institute, Greece, MSc at Cairo University, LLB at Cairo University, and PhD at Mansoura University.
Abstract:
Naturally occurring micron sized silica has gained enormous popularity as a physically active insecticide. Nano-sized silica has insecticidal properties and would be needed in lesser quantity in comparison with conventional insecticides because of the huge surface to volume ratio of nanoparticles. Nano molecules have been widely used in consumer and industrial applications, such as medicine, cosmetics and foods, because they exhibit unique physicochemical properties and innovative functions. However, nanomaterials (NMs) can also be problematic in terms of eliciting a toxicological effect by their small size. The present study was designed to examine the toxic effects of orally administered pesticide Sil-MATRIX 29% (potassium silicate) and silica nano-particles (SiO2-NPs) using male albino rats, at sublethal doses [2/5, 1/4 and 1/8 LD50], relative to control on [body, organs weight such as liver, kidney, heart, spleen, and cytotoxic effect (such as total protein content levels as biochemical aspects)] for 28 and 45 days’ time exposure period. Orally ingested Sil-MATRIX 29% and silica nanoparticles (SiO2-NPs) [2/5, 1/4 and 1/8 LD50] were not associated with significant changes in the average gain of body and organ weight. On the other hand, total protein content value after ingestion with Sil-MATRIX and SiO2-NPs for all doses and treatments time period were increased significantly in a pattern similar to control rats. Our results suggested that the well-dispersed nano-silica cytotoxic effect caused systemic exposure in mouse and induced mutagenic activity. Our information indicated that further studies of relation between physicochemical properties and biological responses are needed for the development and safer form of (NMs).
Jean M François
Université Fédérale Toulouse Midi-Pyrénées, France
Title: Engineering carbon-conserving synthetic pathways for assimilation and conversion of C5/C6 carbon sources into added value chemicals
Biography:
Jean M François got his PhD in Biological Science and Agronomy from the University of Louvain (Belgium) in 1988. He is Professor of Industrial Microbiology and BioNanotechnology at the Federal University of Toulouse, School of Engineer. His research activity concerns integrated physiology and functional genomics in microbial systems, with a specific focus on carbon and energy metabolism in yeast and filamentous fungi . He is author of more than 180 papers and 15 patents and Editor in Chief of BMC Biotechnology for Biofuels.
Abstract:
The development of carbon efficient pathways for added value (bio)chemicals production is the essence of White Biotechnology. The limit of carbon conservation in all (bio)chemical syntheses is determined by the electron balance in substrate(s) and product(s). Frequently, natural metabolic networks do not have the stoichiometric capacity to produce a value-added compound at yields that correspond to the thermodynamic maximum. A good example of natural metabolic networks lacking stoichiometric efficiency is the bioproduction of glycolic acid (GA), a two carbon compound of considerable industrial interest notably in cosmetics and biodegradable polymers. We addressed this objective to approach this maximal conversion yield by employing the following strategies. Firstly, we reconsider a completely different route of C5 assimilation that by-passes the decarboxylation reaction in the pentose phosphate pathway and that rely on the carbon-conserving aldolytic cleavage of X1P or R1P to yield the C2 compound glycolaldehyde and the C3 DHAP compound. This metabolic scheme required the expression of human hexo(fructo)kinase(Khk-C) and human aldolase (Aldo-B). Then glycoaldehyde can be either reduced by endogenous aldehyde reductase to produce ethylene (EG) glycol or oxidized into glycolic acid. With this approach, we obtained yield of EG and GA close to maximal theoretical yield of 1 mol/ mol sugar. Interestingly, we found that the engineered strain expressing this synthetic pathway exhibited a remarkable rewiring of the metabolic networks that culminate with a dramatic reduced metabolites and metabolic energy levels. We then combined this synthetic pathway with the natural glyoxylate shunt that can be engineered to produce GA from DHAP. This combination led to an optimized production strain that produced ~30 % more GA from a xylose/glucose mixture (66%/33%) than when the natural pathway is working alone.