Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

Investigation of the MFS drug transporter family in Candida parapsilosis using CRISPR-Cas9

The function of specific transporters is a key feature underlying drug resistance in Candida species. Drug transporters fall into two main classes – ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS) transporters. Some members of the drug/H (+) antiporter (DHA1) of the MFS superfamily have been shown to function as multidrug transporters. We targeted 16 genes belonging to five families that compose one branch of the DHA1 transporter group. These include MDR1/FLR1, associated with multidrug resistance in C. albicans (3 members); TPO4, associated with polyamine transport (1 member); NAG3/4, associated with transport of N-acetyl glucosamine (2 members); TPO2/3, associated with polyamine transport (1 member); and TPO1/FLU1, possibly associated with fluconazole resistance (9 members). We used CRISPR-Ca9 based gene editing to explore the function of of the five families in C. parapsilosis. All 16 members were individually disrupted by introducing stop codons in the first third of the open reading frames (editing), or by deleting the whole gene. In addition, members of each family were disrupted together, including all 9 members of the TPO1/FLU1 family. CPAR2_603010, CPAR2_207540, and CPAR2_301760 all belonged to the MDR1 family. Editing CPAR2_603010 conferred sensitivity to fluconazole and voriconazole, though disrupting the other two genes had no effect. The azole sensitivity of the CPAR2_603010 edited strain was reverted by introducing the wild type sequence. Disrupting CPAR2_603010 or CPAR2_301760 individually did not affect sensitivity to 4-nitroquinoline 1-oxide. However, the double disruptant was sensitive. Disrupting CPAR2_300760, a member of the TPO1/FLU1 family, resulted in sensitivity to mycophenolic acid. Whole genome sequencing analysis of a strain in which all nine TPO1 genes were disrupted revealed that few off-target effects introduced by the CRISPR-Cas9 system, as few unexpected changes were found after eight rounds of transformation.

A Survey of the Combinatorial Paraphernalia in Protein Synthesis

Statement of the Problem: The combinatorial paraphernalia in protein synthesis to be surveyed are multifarious, embracing, phenomena, processes, activities and materials, all characterized by plurality and dissimilarity. The materials usable are phenomenal and must be a set of discrete plural and dissimilar objects, e.g. the RNA four bases of Adenine, Uracil, Guanine, Cytosine (A,U,G,C) for the activity of permutation for building genetic code. Sequences for protein type sequence composition, proliferation and diversification as inherent in protein synthesis. Methodology and Theoretical Orientation: We are in for combinatorics which is the scientific study of the phenomenon of input/output productivity exhibited by a duality of numeral entities as in permutation of specified set (n) of dissimilar discrete plural. Things and selection (r) of them. The Dalina apparatus of Input/Output Multiplicative Replication system equipped with Square Kinematics View Mixing Technique sourced from inchoate Numeration Science literature being developed by this author is in use for the computation of 4 from 4 permutations of RNA four bases, A,U,G,C constituting the 24 quadruplet genetic code as the workforce in protein synthesis. Findings: The combinatorial paraphernalia in protein synthesis identified and surveyed comprise 14 characteristics, 3 materials and 11 processes/operatives. Conclusion and Significance: The relevance of the several identified and surveyed combinatorial paraphernalia in protein synthesis has been demonstrated by the test of agreeability with the working of the Dalina apparatus of Input/ Output Multiplicative Replication Combinatorial System using the Square Kinematics View Mixing technique for the computation of permutations of RNA four bases A,U,G,C making up the 24 quadruplet genetic code as the workforce in protein synthesis for the substance of all plants and animals throughout CREATION.

AMaLa: Analysis of Directed Evolution Experiments via Annealed Mutational Approximated Landscape

We present Annealed Mutational approximated Landscape (AMaLa), a new method to infer fitness landscapes from Directed Evolution experiments sequencing data. Such experiments typically start from a single wild-type sequence, which undergoes Darwinian in vitro evolution via multiple rounds of mutation and selection for a target phenotype. In the last years, Directed Evolution is emerging as a powerful instrument to probe fitness landscapes under controlled experimental conditions and as a relevant testing ground to develop accurate statistical models and inference algorithms (thanks to high-throughput screening and sequencing). Fitness landscape modeling either uses the enrichment of variants abundances as input, thus requiring the observation of the same variants at different rounds or assuming the last sequenced round as being sampled from an equilibrium distribution. AMaLa aims at effectively leveraging the information encoded in the whole time evolution. To do so, while assuming statistical sampling independence between sequenced rounds, the possible trajectories in sequence space are gauged with a time-dependent statistical weight consisting of two contributions: (i) an energy term accounting for the selection process and (ii) a generalized Jukes–Cantor model for the purely mutational step. This simple scheme enables accurately describing the Directed Evolution dynamics and inferring a fitness landscape that correctly reproduces the measures of the phenotype under selection (e.g., antibiotic drug resistance), notably outperforming widely used inference strategies. In addition, we assess the reliability of AMaLa by showing how the inferred statistical model could be used to predict relevant structural properties of the wild-type sequence.

On iterative methods for bilevel equilibrium problems

We use the notion of Halpern-type sequence recently introduced by the present authors to conclude two strong convergence theorems for solving the bilevel equilibrium problems proposed by Yuying et al. and some authors. Our result excludes some assumptions as were the cases in their results.

Expanding the binding specificity for RNA recognition by a PUF domain

The ability to design a protein to bind specifically to a target RNA enables numerous applications, with the modular architecture of the PUF domain lending itself to new RNA-binding specificities. For each repeat of the Pumilio-1 PUF domain, we generate a library that contains the 8,000 possible combinations of amino acid substitutions at residues critical for RNA contact. We carry out yeast three-hybrid selections with each library against the RNA recognition sequence for Pumilio-1, with any possible base present at the position recognized by the randomized repeat. We use sequencing to score the binding of each variant, identifying many variants with highly repeat-specific interactions. From these data, we generate an RNA binding code specific to each repeat and base. We use this code to design PUF domains against 16 RNAs, and find that some of these domains recognize RNAs with two, three or four changes from the wild type sequence.

NT-CRISPR: Combining natural transformation and CRISPR/Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens

The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for a wide range of projects. To fully harness the potential of this fascinating bacterium, convenient and highly efficient genome editing methods are indispensable to create novel strains, tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This process, called natural transformation, was tamed for genome editing. It displays a high efficiency and is able to mediate uptake of multiple DNA fragments, thereby allowing multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR/Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR/Cas9, targeting the wild type sequence, leading to death of non-edited cells. Through highly efficient cell killing with efficiencies of up to 99.999 %, integration of antibiotic resistance markers became dispensable and thus enabled scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100 % and further demonstrated its applicability for the simultaneous deletion of multiple chromosomal regions. Lastly, we demonstrated that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby massively broadens the target spectrum.

AMaLa: analysis of Directed Evolution experiments via Annealed Mutational approximated Landscape

We present Annealed Mutational approximated landscape (AMaLa), a new method to infer fitness landscapes from Directed Evolution experiment sequencing data. Directed Evolution experiments typically start from a single wild-type sequence, which undergoes Darwinian in vitro evolution acted via multiple rounds of mutation and selection with respect to a target phenotype. In the last years, Directed Evolution is emerging as a powerful instrument to probe fitness landscapes under controlled experimental condition and, thanks to the use of high-throughput sequencing of the different rounds, as a relevant testing ground to develop accurate statistical models and inference algorithms. Fitness landscape modeling strategies, either use as input data the enrichment of variants abundances and hence require observing the same variants at different rounds, or they simply assume that the variants at the last sequenced round are the results of a sampling process at equilibrium. AMaLa aims at leveraging effectively the information encoded in the time evolution of all sequenced rounds. To do so, on the one hand we assume statistical sampling independence between sequenced rounds, and on the other we gauge all possible trajectories in sequence space with a time-dependent statistical weight consisting of two contributions: (i) a statistical energy term accounting for the selection process, (ii) a simple generalized Jukes-Cantor model to describe the purely mutational step. This simple scheme allows us to accurately describe the Directed Evolution dynamics in a concrete experimental setup and to infer a fitness landscape that reproduces correctly the measures of the phenotype under selection (e.g. antibiotic drug resistance), notably outperforming widely used inference strategies. We assess the reliability of AMaLa by showing how the inferred statistical model could be used to predict relevant structural properties of the wild-type sequence, and to reproduce the mutational effects of large scale functional screening not used to train the model.

Substantiation of process schemes of field development taking into account technogenic conversion

Introduction. Mining disturbs the natural state of the rock mass. Besides, the field reserves are of a different degree and nature of man-induced changes, which directly affects mining prime cost and ore marketable value. The scientific and practical relevance of the research consists in ensuring increased completeness and economic efficiency of field development by applying the development systems of different classes and taking into account the man-induced changes in reserves in mining dynamics. Research aim is to substantiate the technological schemes of mining, that take into account the possibility and feasibility of developing the reserves which have been affected by the man-induced changes, in particular when switching from one class of development systems to another. Research methods. In this research, based on the analysis of the mining and geological conditions of copper sulfide deposits, economic and mathematical modeling of developing an ore deposit by development systems of different classes was carried out, and the man-induced changes in underground reserves in the process of mining were taken into account. Results. Based on the modeling results, it was found that the increase in the economic efficiency of the deposit development is provided by justifying the type, sequence, and limits of applying different geotechnologies related to different classes of development systems within the entire deposit and a separate ore body. Conclusion. The research results can be used when designing fields in terms of selecting and substantiating the applied development systems.