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

Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides

RNA secondary structure prediction using free energy minimization is one method to gain an approximation of structure. Constraints generated by enzymatic mapping or chemical modification can improve the accuracy of secondary structure prediction. We report a facile method that identifies single-stranded regions in RNA using short, randomized DNA oligonucleotides and RNase H cleavage. These regions are then used as constraints in secondary structure prediction. This method was used to improve the secondary structure prediction of Escherichia coli 5S rRNA. The lowest free energy structure without constraints has only 27% of the base pairs present in the phylogenetic structure. The addition of constraints from RNase H cleavage improves the prediction to 100% of base pairs. The same method was used to generate secondary structure constraints for yeast tRNAPhe, which is accurately predicted in the absence of constraints (95%). Although RNase H mapping does not improve secondary structure prediction, it does eliminate all other suboptimal structures predicted within 10% of the lowest free energy structure. The method is advantageous over other single-stranded nucleases since RNase H is functional in physiological conditions. Moreover, it can be used for any RNA to identify accessible binding sites for oligonucleotides or small molecules

Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells

One of the critical steps that governs the inhibitory effect of antisense RNA on target gene expression is the association of the antisense RNA with the target RNA molecules. However, until now, no systematic method has been available to select the suitable parts of a gene as antisense targets. In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5′ splice site (5′ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target. The 5′ free end of U1snRNA was replaced with the antisense sequence against the K-ras gene to generate a hyperstable U1snRNA, whose binding stability to 5′ss of the K-ras transcript is ten-fold higher than that of wild-type U1snRNA. The efficacy of such hyperstable U1snRNA was examined by transducing the expression plasmids into human pancreatic cancer cell lines. This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls. Furthermore, hyperstable U1snRNA suppressed intraperitoneal dissemination of pancreatic cancer cells in vivo. Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells

An RNA toolbox for single-molecule force spectroscopy studies

Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one's disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs