New glucose isomerase - fit for biorefinery challenge

Biofuel is not the bio-product with the highest value-addition, especially with the currently low oil price in mind. Modern concepts of biorefineries therefore tend to include production of more valuable products than ethanol, such as bioplastics. Hydroxymethyl furfural (HMF) is considered to be a central platform chemical for biomaterials production. HMF can be produced from hexose sugars, however the conversion is much more efficient ketoses ketoses such fructose than with aldehydes (glucose, mannose and so on). Glucose is the only sufficiently abandoned sugar monomer to potentially become a row material for commodity chemicals manufacturing, wood being the most likely source of glucose. Glucose can be then enzymatically converted to its ketose isomer – fructose using xylose (glucose) isomerase. The glucose isomerases are widely commercially available: they are one of the largest in volume in the industrial enzyme market for their production of widely-used High Fructose Syrups (HFS) for food applications. However, the currently available commercial enzymes are highly sensitive to the substrate sugar purity, which is well acceptable in food industry application. Typically, even sugar produced from starch requires activated carbon filtration, ion exchange chromatography and degasification before it can proceed to isomerization reaction. Sugars produced in 2nd generation biorefinery (especially from wood) have much more impurities than starch derived sugar, including lignin, extractives, etc, and required level of purity is not justified for the technical sugar. Taking this challenge, we set to develop and industrial glucose isomerase that can work directly in lignocellulosic biomass hydrolysate. To address the choice of enzyme prototypes covering most structurally diverse groups, we obtained a custom made 3dm database (ordered from Bio-Prodict BV, Netherlands). The database contained around 25 000 protein sequences from public databases aligned and uniformly numbered based on structural alignment or strong homology. Database, where annotation of proteins was not taken in account while building it, eventually contained xylose isomerases L-rhamnose isomerases, hydropiruvate isomerase, innosose isomerases, D-tagatose epimerases, L-ribulose-phosphate epimerases, mannonate dehydratases and endonucleases. This tool gave a general view on structural diversity of known characterized and just annotated xylose isomerase, and helped to find a representative pool of prototypes to test for our special requirements. Among over 20 tested candidates, we found a new extremely robust enzyme, which outperformed every reference enzyme in glucose isomerization in crude lignocellulosic hydrolysate. The broad scope of proteins represented in 3dm database allowed unprecedented opportunity to analyze the proteins sharing the same fold in terms of what makes them functionally distinct. Focusing our attention on xylose isomerases, we were able to identify several positions, which make a protein with such fold a xylose isomerase. Some of those positions have never been mentioned in the literature as mutational hot spots or as residues essential for the function. We constructed focused libraries with variation in these positions and were able to find enzyme variants with strongly altered substrate preferences between glucose and fructose and enabled doubling the efficiency of glucose isomerization by the new enzyme. The enzyme production in E.coli was scaled up to industrial scale. Thus using bioinformatics approach combined with protein engineering, we developed an industrial enzyme that enables sugar valorization and platform chemicals production in biorefinery streamline. This work was supported by EU via Horizon 2020 projects RETAPP, and BIOFOREVER

Enzymes – Key Elements of the Future Biorefineries

The biorefinery concept in its modern meaning has emerged after it has become apparent that biofuel production from non-food biomass is struggling for economic viability. Lignocellulosic biomass is more recalcitrant and more complex than the starch-based feedstocks used for food. The former, therefore, calls for a more complex approach to its utilization. This chapter reflects MetGen’s vision of the future development of biorefineries. We will discuss the zero-waste approach to lignocellulosic biomass utilization and various ways to valorize the resulting streams to boost the economic viability of the biorefinery. We will mostly explore the relevant enzyme-based approaches and will make a special focus on lignin valorization. Enzymatic and cell-based approaches to sugar valorization will be discussed as well

Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms

VECTORS, CONTAINING THE ENHANGER OF TRANSLATION FOR THE EXPRESSION OF ARTIFICIAL GENES IN E.COLI

The object of investigation: the genes of mature interleukine-1 alpha of the man and receptor antagonists of the interleukine-1 alpha of the man. The purpose of the work: creating the effectivevectors for the prokaryotic expression of the artificial genes, and obtaining on its base, of the strains-producers of interleukine-1 alpha of the man and its receptor antagonists. The interinfluence of the direct and conjugate translation in the obtained two-cistron structure has been investigated. The strains-superproducers of proteins of interleukine-1 alpha of the man and its receptor antagonist have been obtained. The field of application: the molecular biology, the biotechnology, the medicineAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase)

Enzymatic Processes to Unlock the Lignin Value

Main hurdles of lignin valorization are its diverse chemical composition, recalcitrance, and poor solubility due to high-molecular weight and branched structure. Controlled fragmentation of lignin could lead to its use in higher value products such as binders, coatings, fillers, etc. Oxidative enzymes (i.e., laccases and peroxidases) have long been proposed as a potentially promising tool in lignin depolymerization. However, their application was limited to ambient pH, where lignin is poorly soluble in water. A Finnish biotechnology company, MetGen Oy, that designs and supplies industrial enzymes, has developed and brought to market several lignin oxidizing enzymes, including an extremely alkaline lignin oxidase MetZyme® LIGNO™, a genetically engineered laccase of bacterial origin. This enzyme can function at pH values as high as 10–11 and at elevated temperatures, addressing lignin at its soluble state. In this article, main characteristics of this enzyme as well as its action on bulk lignin coming from an industrial process are demonstrated. Lignin modification by MetZyme® LIGNO™ was characterized by size exclusion chromatography, UV spectroscopy, and dynamic light scattering for monitoring particle size of solubilized lignin. Under highly alkaline conditions, laccase treatment not only decreased molecular weight of lignin but also increased its solubility in water and altered its dispersion properties. Importantly, organic solvent-free soluble lignin fragmentation allowed for robust industrially relevant membrane separation technologies to be applicable for product fractionation. These enzyme-based solutions open new opportunities for biorefinery lignin valorization thus paving the way for economically viable biorefinery business

Analysis of the reduction of TurboGFP levels by antisense mimic oligomers in Phoenix Eco cells transfected with p2FP-RNAi vector

<p><b>Copyright information:</b></p><p>Taken from "Hydroxyproline-based DNA mimics provide an efficient gene silencing and "</p><p>Nucleic Acids Research 2006;34(8):2247-2257.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456331.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Co-transfection of oligomers was performed in the presence of LFA. The cells transformed with the vector alone were analyzed as controls. () Sequences of oligomers designed to target the translational start site of the TurboGFP mRNA. () Analysis of green and red fluorescence intensity of the cells treated with 0.5 µM oligomers, or with 0.2 µM dsRNA, after 24 h. The average data of three separate experiments are shown. () Fluorescent microscopy images of the cells transfected with p2FP-RNAi and treated with 0.5 µM mimic oligomer samples for 16 and 24 h. Panels show examples of cells treated with 0.2 µM dsRNA (a); mismatched pHypNA oligomer (b); duplex of antisense pHypNA oligomer with CT-ODN (c); duplex of antisense HypNA-pPNA oligomer with CT-ODN (d); antisense pHypNA oligomer (e) and antisense HypNA-pPNA oligomer (f). Panel (g) shows cells untreated with oligomers. () Effect of the antisense oligomer concentration on the TurboGFP production in cells. Fluorescence was measured 24 h after the transfection. Average data points from three independent experiments are shown

Images of tadpole heads as seen from the dorsal side and the sequences of MO, pHypNA and HypNA-pPNA oligomers used in mRNA translation inhibitory assays

<p><b>Copyright information:</b></p><p>Taken from "Hydroxyproline-based DNA mimics provide an efficient gene silencing and "</p><p>Nucleic Acids Research 2006;34(8):2247-2257.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456331.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The injection of antisense oligomers results in severe abnormalities of the head structure. The side of injection is shown by red arrow. () A control tadpole head; ( and ) heads of tadpoles developed from the embryos microinjected with the antisense MO, pHypNA and HypNA-pPNA oligomers, respectively; ( and ) heads of tadpoles developed from the embryos microinjected with the mismatched pHypNA and HypNA-pPNA oligomers

Cellular uptake and intracellular distribution of the fluorescein-labeled mimic oligomers (TCACTCAACACTCAC-Flu)

<p><b>Copyright information:</b></p><p>Taken from "Hydroxyproline-based DNA mimics provide an efficient gene silencing and "</p><p>Nucleic Acids Research 2006;34(8):2247-2257.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456331.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Fluorescent analysis of the delivery of oligomers (1 µM) into Phoenix Eco cells performed 20 h after the treatment under various delivery conditions: in the presence, or in the absence, of LFA, 6 mM CaCl, and 100 µM chloroquine (ClQ). () Confocal microscopy images of the pHypNA oligomer uptake after 20 h incubated with unfixed Phoenix Eco cells in the presence of: LFA (1); LFA/6mM Ca (2); CT-ODN/LFA (3) and LFA/100 µM ClQ (4)