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tRNA engineering for manipulating genetic code.
Katoh, T, Iwane, Y, Suga, H
RNA biology. 2018;(4-5):453-460
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Abstract
In ribosomal translation, only 20 kinds of proteinogenic amino acids (pAAs), namely 19 l-amino acids and glycine, are exclusively incorporated into polypeptide chain. To overcome this limitation, various methods to introduce non-proteinogenic amino acids (npAAs) other than the 20 pAAs have been developed to date. However, the repertoire of amino acids that can be simultaneously introduced is still limited. Moreover, the efficiency of npAA incorporation is not always sufficient depending on their structures. Fidelity of translation is sometimes low due to misincorporation of competing pAAs and/or undesired translation termination. Here, we provide an overview of efforts to solve these issues, focusing on the engineering of tRNAs.
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Investigating Potassium Channels in Budding Yeast: A Genetic Sandbox.
Mackie, TD, Brodsky, JL
Genetics. 2018;(3):637-650
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Abstract
Like all species, the model eukaryote Saccharomyces cerevisiae, or Bakers' yeast, concentrates potassium in the cytosol as an electrogenic osmolyte and enzyme cofactor. Yeast are capable of robust growth on a wide variety of potassium concentrations, ranging from 10 µM to 2.5 M, due to the presence of a high-affinity potassium uptake system and a battery of cation exchange transporters. Genetic perturbation of either of these systems retards yeast growth on low or high potassium, respectively. However, these potassium-sensitized yeast are a powerful genetic tool, which has been leveraged for diverse studies. Notably, the potassium-sensitive cells can be transformed with plasmids encoding potassium channels from bacteria, plants, or mammals, and subsequent changes in growth rate have been found to correlate with the activity of the introduced potassium channel. Discoveries arising from the use of this assay over the past three decades have increased our understanding of the structure-function relationships of various potassium channels, the mechanisms underlying the regulation of potassium channel function and trafficking, and the chemical basis of potassium channel modulation. In this article, we provide an overview of the major genetic tools used to study potassium channels in S. cerevisiae, a survey of seminal studies utilizing these tools, and a prospective for the future use of this elegant genetic approach.
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Nanoparticle-Mediated Delivery towards Advancing Plant Genetic Engineering.
Cunningham, FJ, Goh, NS, Demirer, GS, Matos, JL, Landry, MP
Trends in biotechnology. 2018;(9):882-897
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Abstract
Genetic engineering of plants has enhanced crop productivity in the face of climate change and a growing global population by conferring desirable genetic traits to agricultural crops. Efficient genetic transformation in plants remains a challenge due to the cell wall, a barrier to exogenous biomolecule delivery. Conventional delivery methods are inefficient, damaging to tissue, or are only effective in a limited number of plant species. Nanoparticles are promising materials for biomolecule delivery, owing to their ability to traverse plant cell walls without external force and highly tunable physicochemical properties for diverse cargo conjugation and broad host range applicability. With the advent of engineered nuclease biotechnologies, we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering.
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4.
tRNAPyl: Structure, function, and applications.
Tharp, JM, Ehnbom, A, Liu, WR
RNA biology. 2018;(4-5):441-452
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Abstract
Pyrrolysine is the 22nd proteinogenic amino acid encoded into proteins in response to amber (TAG) codons in a small number of archaea and bacteria. The incorporation of pyrrolysine is facilitated by a specialized aminoacyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNAPyl). The secondary structure of tRNAPyl contains several unique features not found in canonical tRNAs. Numerous studies have demonstrated that the PylRS/tRNAPyl pair from archaea is orthogonal in E. coli and eukaryotic hosts, which has led to the widespread use of this pair for the genetic incorporation of non-canonical amino acids. In this brief review we examine the work that has been done to elucidate the structure of tRNAPyl, its interaction with PylRS, and survey recent progress on the use of tRNAPyl as a tool for genetic code expansion.
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5.
The Pragmatic Introduction and Expression of Microbial Transgenes in Plants.
Ali, S, Park, SK, Kim, WC
Journal of microbiology and biotechnology. 2018;(12):1955-1970
Abstract
Several genetic strategies have been proposed for the successful transformation and expression of microbial transgenes in model and crop plants. Here, we bring into focus the prominent applications of microbial transgenes in plants for the development of disease resistance; mitigation of stress conditions; augmentation of food quality; and use of plants as "bioreactors" for the production of recombinant proteins, industrially important enzymes, vaccines, antimicrobial compounds, and other valuable secondary metabolites. We discuss the applicable and cost-effective approaches of transgenesis in different plants, as well as the limitations thereof. We subsequently present the contemporary developments in targeted genome editing systems that have facilitated the process of genetic modification and manifested stable and consumer-friendly genetically modified plants and their products. Finally, this article presents the different approaches and demonstrates the introduction and expression of microbial transgenes for the improvement of plant resistance to pathogens and abiotic stress conditions and the production of valuable compounds, together with the promising research progress in targeted genome editing technology. We include a special discussion on the highly efficient CRISPR-Cas system helpful in microbial transgene editing in plants.
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Concerns regarding 'off-target' activity of genome editing endonucleases.
Kadam, US, Shelake, RM, Chavhan, RL, Suprasanna, P
Plant physiology and biochemistry : PPB. 2018;:22-30
Abstract
Genome editing (GE) tools ensure targeted mutagenesis and sequence-specific modification in plants using a wide resource of customized endonucleases; namely, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system. Among these, in recent times CRISPR/Cas9 has been widely used in functional genomics and plant genetic modification. A significant concern in the application of GE tools is the occurrence of 'off-target' activity and induced mutations, which may impede functional analysis and gene activity studies. Moreover, the 'off-target' activity results in either not reported or unknown, difficult to detect, produce non-quantifiable cellular signaling and physiological effects. In the past few years, several experimental methods have been developed to identify undesired mutations and to curtail 'off-target' cleavage. Improvement in target specificity and minimizing 'off-target' activity will offer better applications of GE technology in plant biology and crop improvement.
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Prospects of genetic engineering utilizing potential genes for regulating arsenic accumulation in plants.
Kumari, P, Rastogi, A, Shukla, A, Srivastava, S, Yadav, S
Chemosphere. 2018;:397-406
Abstract
The rapid pace of industrial, agricultural and anthropogenic activities in the 20th century has resulted in contamination of heavy metals across the globe. Arsenic (As) is a ubiquitous, naturally occurring toxic metalloid, contaminating the soil and water and affecting human health in several countries. Several physicochemical methods exist for the cleanup of As contamination but these are expensive and disastrous to microbes and soil. Plant based remediation approaches are low cost and environmentally safe. Hence, extensive biochemical, molecular and genetic experiments have been conducted to understand plants' responses to As stress and have led to the identification of potential genes. The available knowledge needs to be utilized to either reduce As accumulation in crop plants (rice) or to enhance As levels in shoots of hyperaccumulators (Pteris vittata). Gene manipulation using biotechnological tools can be an effective approach to exploit the potential genes (plasmamembrane and vacuolar transporters, glutathione and phytochelatin biosynthetic enzymes, etc.) playing pivotal roles in uptake, translocation, transformation, complexation, and compartmentalization of As in plants. The transgenic plants with increased tolerance to As and altered (increased/decreased) As accumulation have been developed. The need, however, exists to design plants with altered expression of two or more genes for harmonizing various events (like arsenate reduction, arsenite complexation, sequestration and translocation) so as to achieve desirable reduction (crop plants) or increase (phytoremediator plants) in As content. This review sheds light on transgenic approaches adopted to modulate As levels in plants and proposes future directions to achieve desirable results.
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Recent advances in reconstructing microbial secondary metabolites biosynthesis in Aspergillus spp.
He, Y, Wang, B, Chen, W, Cox, RJ, He, J, Chen, F
Biotechnology advances. 2018;(3):739-783
Abstract
High throughput genome sequencing has revealed a multitude of potential secondary metabolites biosynthetic pathways that remain cryptic. Pathway reconstruction coupled with genetic engineering via heterologous expression enables discovery of novel compounds, elucidation of biosynthetic pathways, and optimization of product yields. Apart from Escherichia coli and yeast, fungi, especially Aspergillus spp., are well known and efficient heterologous hosts. This review summarizes recent advances in heterologous expression of microbial secondary metabolite biosynthesis in Aspergillus spp. We also discuss the technological challenges and successes in regard to heterologous host selection and DNA assembly behind the reconstruction of microbial secondary metabolite biosynthesis.
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Enhancing the performance of brewing yeasts.
Karabín, M, Jelínek, L, Kotrba, P, Cejnar, R, Dostálek, P
Biotechnology advances. 2018;(3):691-706
Abstract
Beer production is one of the oldest known traditional biotechnological processes, but is nowadays facing increasing demands not only for enhanced product quality, but also for improved production economics. Targeted genetic modification of a yeast strain is one way to increase beer quality and to improve the economics of beer production. In this review we will present current knowledge on traditional approaches for improving brewing strains and for rational metabolic engineering. These research efforts will, in the near future, lead to the development of a wider range of industrial strains that should increase the diversity of commercial beers.
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10.
Challenges of site-specific selenocysteine incorporation into proteins by Escherichia coli.
Fu, X, Söll, D, Sevostyanova, A
RNA biology. 2018;(4-5):461-470
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Abstract
Selenocysteine (Sec), a rare genetically encoded amino acid with unusual chemical properties, is of great interest for protein engineering. Sec is synthesized on its cognate tRNA (tRNASec) by the concerted action of several enzymes. While all other aminoacyl-tRNAs are delivered to the ribosome by the elongation factor Tu (EF-Tu), Sec-tRNASec requires a dedicated factor, SelB. Incorporation of Sec into protein requires recoding of the stop codon UGA aided by a specific mRNA structure, the SECIS element. This unusual biogenesis restricts the use of Sec in recombinant proteins, limiting our ability to study the properties of selenoproteins. Several methods are currently available for the synthesis selenoproteins. Here we focus on strategies for in vivo Sec insertion at any position(s) within a recombinant protein in a SECIS-independent manner: (i) engineering of tRNASec for use by EF-Tu without the SECIS requirement, and (ii) design of a SECIS-independent SelB route.