Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches.

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

GTP-binding protein Era: a novel gene target for biofuel production.

BACKGROUND: Biodiesel production using cyanobacteria is a promising alternative to fossil fuels. In this study we created a transposon library of Synechococcus elongatus PCC 7942 in order to identify novel gene targets for enhanced fatty acid and hydrocarbon production. The transposon library was subsequently screened for desirable traits using macro- and microscopic observations as well as staining with the lipophilic dye Nile Red. RESULTS: Based on the screening results, we selected a single mutant, which has an insertion in the gene encoding for the GTP-binding protein Era. We subsequently verified the phenotype-genotype relation by overexpression, reintroducing and complementing the mutation. Overexpression of era caused a reduction in the cell size in the late exponential phase of growth and an increase in the total amount of intracellular fatty acids. Reintroduction of the inactivated transposon caused a significant increase in the cellular length as well as changes in the amounts of individual hydrocarbons and fatty acids. Ectopic complementation of this mutation fully restored the hydrocarbon production profile to that of wild-type and partially restored the fatty acid production. Moreover, the cellular size was significantly smaller than that of the inactivated transposon mutant. CONCLUSIONS: The GTP-binding protein Era has never been studied in cyanobacteria and proved to be an essential gene for S. elongatus PCC 7942. We also found that this protein is important for hydrocarbon and fatty acid metabolism as well as determination of the cell size in PCC 7942. Our results suggest that the GTP-binding protein Era can be used as a novel target for further improvement of biofuel precursors production.

High throughput AS LNA qPCR method for the detection of a specific mutation in poliovirus vaccine strains.

Sabin Inactivated Poliovirus Vaccine (sIPV) has become one of the preferred vaccination options for the last step in the Poliovirus eradication program. Sequencing of poliovirus samples is needed during the manufacturing of poliovirus vaccines to assure the safety and immunogenicity of these vaccines. Next-generation sequencing analysis is the current costly and time-consuming gold standard for monitoring the manufacturing processes. We developed a low-cost and quick, highly sensitive, and allele-specific locked nucleic acid-probe-based reverse transcription quantitative PCR alternative that can accurately detect mutations in poliovirus vaccine samples during process development, scaling up, and release. Using the frequently in vitro occurring and viral replication-impacting VP1-E295K mutation as a showcase, we show that this technology can accurately detect E295K mutations in poliovirus 2 samples to similar levels as NGS. The qPCR technology was developed employing a synthetic dsDNA fragment-based standard curve containing mixes of E295K-WT (wildtype) and Mut (mutant) synthetic dsDNA fragments ranging from 1 × 107 copies/µL to 1 × 102 copies/µL to achieve a linear correlation with R2 > 0.999, and PCR efficiencies of 95-105 %. Individual standard concentration levels achieved accuracies of ≥92 % (average 96 %) and precisions of ≤17 % (average 3.3 %) RSD. Specificity of locked nucleic acid (LNA)-probes was confirmed in the presence and absence of co-mutations in the probe-binding region. Application of the developed assay to Sabin Poliovirus type 2 production run samples, illustrated a linear relationship with an R2 of 0.994, and an average accuracy of 97.2 % of the variant (allele)-specific AS LNA qPCR result, compared to NGS. The assay showed good sensitivity for poliovirus samples, containing E295K mutation levels between 0 % and 95 % (quantification range). In conclusion, the developed AS LNA qPCR presents a valuable low-cost, and fast tool, suitable for the process development and quality control of polio vaccines.

Phytohormone Production by the Endophyte Bacillus safensis TS3 Increases Plant Yield and Alleviates Salt Stress.

Endophytic bacteria can be used to overcome the effect of salinity stress and promote plant growth and nutrient uptake. Bacillus safensis colonizes a wide range of habitats due to survival in extreme environments and unique physiological characteristics, such as a high tolerance for salt, heavy metals, and ultraviolet and gamma radiations. The aim of our study was to examine the salt resistance of the endophytic strain TS3 B. safensis and its ability to produce phytohormones and verify its effect on plant yield in field trials and the alleviation of salt stress in pot experiments. We demonstrate that the strain TS3 is capable of producing enzymes and phytohormones such as IAA, ABA and tZ. In pot experiments with radish and oat plants in salinization, the strain TS3 contributed to the partial removal of the negative effect of salinization. The compensatory effect of the strain TS3 on radish plants during salinization was 46.7%, and for oats, it was 108%. We suppose that such a pronounced effect on the plants grown and the salt stress is connected with its ability to produce phytohormones. Genome analysis of the strain TS3 showed the presence of the necessary genes for the synthesis of compounds responsible for the alleviation of the salt stress. Strain B. safensis TS3 can be considered a promising candidate for developing biofertilizer to alleviate salt stress and increase plant yield.

Draft Genome Sequence of the Tomato Stem Endophyte Bacillus safensis TS3.

Tomato stem endophyte Bacillus safensis TS3 was isolated from surface-sterilized stems of greenhouse tomato plants. Here, we sequenced the complete genome of this strain to understand the molecular mechanisms underlying its beneficial activities.

Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering.

The need to develop and improve sustainable energy resources is of eminent importance due to the finite nature of our fossil fuels. This review paper deals with a third generation renewable energy resource which does not compete with our food resources, cyanobacteria. We discuss the current state of the art in developing different types of bioenergy (ethanol, biodiesel, hydrogen, etc.) from cyanobacteria. The major important biochemical pathways in cyanobacteria are highlighted, and the possibility to influence these pathways to improve the production of specific types of energy forms the major part of this review.

Draft Genome Sequence of Plant Growth-Promoting Bacillus velezensis BS89.

The plant growth-promoting bacterium Bacillus velezensis BS89 was isolated from the rhizosphere of winter wheat. Strain BS89 has the ability to promote plant growth and produce a mix of auxins and vitamins. Here, we sequenced the complete genome of this strain to understand the molecular mechanisms underlying its beneficial activities.

Carbohydrate Binding Modules: Diversity of Domain Architecture in Amylases and Cellulases From Filamentous Microorganisms.

Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of Streptomyces and Aspergillus that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.

Profile Comparer Extended: phylogeny of lytic polysaccharide monooxygenase families using profile hidden Markov model alignments.

Insight into the inter- and intra-family relationship of protein families is important, since it can aid understanding of substrate specificity evolution and assign putative functions to proteins with unknown function. To study both these inter- and intra-family relationships, the ability to build phylogenetic trees using the most sensitive sequence similarity search methods (e.g. profile hidden Markov model (pHMM)-pHMM alignments) is required. However, existing solutions require a very long calculation time to obtain the phylogenetic tree. Therefore, a faster protocol is required to make this approach efficient for research. To contribute to this goal, we extended the original Profile Comparer program (PRC) for the construction of large pHMM phylogenetic trees at speeds several orders of magnitude faster compared to pHMM-tree. As an example, PRC Extended (PRCx) was used to study the phylogeny of over 10,000 sequences of lytic polysaccharide monooxygenase (LPMO) from over seven families. Using the newly developed program we were able to reveal previously unknown homologs of LPMOs, namely the PFAM Egh16-like family. Moreover, we show that the substrate specificities have evolved independently several times within the LPMO superfamily. Furthermore, the LPMO phylogenetic tree, does not seem to follow taxonomy-based classification.

The discovery of novel LPMO families with a new Hidden Markov model.

BACKGROUND: Renewable biopolymers, such as cellulose, starch and chitin are highly resistance to enzymatic degradation. Therefore, there is a need to upgrade current degradation processes by including novel enzymes. Lytic polysaccharide mono-oxygenases (LPMOs) can disrupt recalcitrant biopolymers, thereby enhancing hydrolysis by conventional enzymes. However, novel LPMO families are difficult to identify using existing methods. Therefore, we developed a novel profile Hidden Markov model (HMM) and used it to mine genomes of ascomycetous fungi for novel LPMOs. RESULTS: We constructed a structural alignment and verified that the alignment was correct. In the alignment we identified several known conserved features, such as the histidine brace and the N/Q/E-X-F/Y motif and previously unidentified conserved proline and glycine residues. These residues are distal from the active site, suggesting a role in structure rather than activity. The multiple protein alignment was subsequently used to build a profile Hidden Markov model. This model was initially tested on manually curated datasets and proved to be both sensitive (no false negatives) and specific (no false positives). In some of the genomes analyzed we identified a yet unknown LPMO family. This new family is mostly confined to the phyla of Ascomycota and Basidiomycota and the class of Oomycota. Genomic clustering indicated that at least some members might be involved in the degradation of ß-glucans, while transcriptomic data suggested that others are possibly involved in the degradation of pectin. CONCLUSIONS: The newly developed profile hidden Markov Model was successfully used to mine fungal genomes for a novel family of LPMOs. However, the model is not limited to bacterial and fungal genomes. This is illustrated by the fact that the model was also able to identify another new LPMO family in Drosophila melanogaster. Furthermore, the Hidden Markov model was used to verify the more distant blast hits from the new fungal family of LPMOs, which belong to the Bivalves, Stony corals and Sea anemones. So this Hidden Markov model (Additional file 3) will help the broader scientific community in identifying other yet unknown LPMOs.