scholarly journals Targeted genome editing of plants and plant cells for biomanufacturing

2021 ◽  
Author(s):  
J. F. Buyel ◽  
E. Stöger ◽  
L. Bortesi
Keyword(s):  
Genome Editing ◽  
Genetic Manipulation ◽  
Molecular Farming ◽  
Plant Cells ◽  
Building Blocks ◽  
General Purpose ◽  
Site Directed Mutagenesis ◽  
Limited Information ◽  
Case Examples ◽  
Cost Structures

AbstractPlants have provided humans with useful products since antiquity, but in the last 30 years they have also been developed as production platforms for small molecules and recombinant proteins. This initially niche area has blossomed with the growth of the global bioeconomy, and now includes chemical building blocks, polymers and renewable energy. All these applications can be described as “plant molecular farming” (PMF). Despite its potential to increase the sustainability of biologics manufacturing, PMF has yet to be embraced broadly by industry. This reflects a combination of regulatory uncertainty, limited information on process cost structures, and the absence of trained staff and suitable manufacturing capacity. However, the limited adaptation of plants and plant cells to the requirements of industry-scale manufacturing is an equally important hurdle. For example, the targeted genetic manipulation of yeast has been common practice since the 1980s, whereas reliable site-directed mutagenesis in most plants has only become available with the advent of CRISPR/Cas9 and similar genome editing technologies since around 2010. Here we summarize the applications of new genetic engineering technologies to improve plants as biomanufacturing platforms. We start by identifying current bottlenecks in manufacturing, then illustrate the progress that has already been made and discuss the potential for improvement at the molecular, cellular and organism levels. We discuss the effects of metabolic optimization, adaptation of the endomembrane system, modified glycosylation profiles, programmable growth and senescence, protease inactivation, and the expression of enzymes that promote biodegradation. We outline strategies to achieve these modifications by targeted gene modification, considering case-by-case examples of individual improvements and the combined modifications needed to generate a new general-purpose “chassis” for PMF.

scholarly journals Flavin Adenine Dinucleotide-Dependent Halogenase XanH and Engineering of Multifunctional Fusion Halogenases

2020 ◽  
Vol 86 (18) ◽  
Author(s):  
Lingxin Kong ◽  
Qing Wang ◽  
Zixin Deng ◽  
Delin You
Keyword(s):  
Protein Engineering ◽  
Fusion Protein ◽  
Natural Product ◽  
Flavin Adenine Dinucleotide ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Good Alternative ◽  
Limited Information ◽  
Flavin Reductase ◽  
Free Standing

ABSTRACT Xantholipin (compound 1), a polycyclic xanthone antibiotic, exhibited strong antibacterial activities and showed potent cytotoxicity. The biosynthetic gene cluster of compound 1 has been identified in our previous work, and the construction of xanthone nucleus has been well demonstrated. However, limited information of the halogenation involved in compound 1 biosynthesis is available. In this study, based on the genetic manipulation and biochemical assay, we characterized XanH as an indispensable flavin adenine dinucleotide (FAD)-dependent halogenase (FDH) for the biosynthesis of compound 1. XanH was found to be a bifunctional protein capable of flavin reduction and chlorination and exclusively used the NADH. However, the reduced flavin could not be fully and effectively utilized, and the presence of an extra flavin reductase (FDR) and chemical-reducing agent could promote the halogenation. XanH accepted its natural free-standing substrate with angular fused polycyclic aromatic systems. Meanwhile, it exhibited moderate halogenation activity and possessed high substrate specificity. The requirement of extra FDR for higher halogenation activity is tedious for future engineering. To facilitate efforts in engineering XanH derivative proteins, we constructed the self-sufficient FDR-XanH fusion proteins. The fusion protein E1 with comparable activities to that of XanH could be used as a good alternative for future protein engineering. Taken together, these findings reported here not only improve the understanding of polycyclic xanthones biosynthesis but also expand the substrate scope of FDH and pave the way for future engineering of biocatalysts for new active substance synthesis. IMPORTANCE Halogenation is important in medicinal chemistry and plays an essential role in the biosynthesis of active secondary metabolites. Halogenases have evolved to catalyze reactions with high efficiency and selectivity, and engineering efforts have been made to engage the selective reactivity in natural product biosynthesis. The enzymatic halogenations are an environmentally friendly approach with high regio- and stereoselectivity, which make it a potential complement to organic synthesis. FDHs constitute one of the most extensively elucidated class of halogenases; however, the inventory awaits to be expanded for biotechnology applications and for the generation of halogenated natural product analogues. In this study, XanH was found to reduce flavin and halogenated the freely diffusing natural substrate with an angular fused hexacyclic scaffold, findings which were different from those for the exclusively studied FDHs. Moreover, the FDR-XanH fusion protein E1 with comparable reactivity to that of XanH serves as a successful example of genetic fusions and sets an important stage for future protein engineering.

scholarly journals Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1526
Author(s):  
Joanna E. Kowalczyk ◽  
Shreya Saha ◽  
Miia R. Mäkelä
Keyword(s):  
Genome Editing ◽  
Plant Biomass ◽  
Genetic Manipulation ◽  
White Rot ◽  
White Rot Fungus ◽  
Detailed Knowledge ◽  
Wild Type ◽  
Low Frequencies ◽  
Dichomitus Squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

scholarly journals Biodiversity Literature Repository: Building the customized FAIR repository by using custom metadata

2021 ◽  
Vol 5 ◽  
Author(s):  
Alexandros Ioannidis-Pantopikos ◽  
Donat Agosti
Keyword(s):  
Particle Physics ◽  
Building Blocks ◽  
General Purpose ◽  
Digital Object Identifier ◽  
Global Biodiversity Information Facility ◽  
Local Networks ◽  
Representational State Transfer ◽  
Processing Pipeline ◽  
European Laboratory ◽  
Machine Readable

In the landscape of general-purpose repositories, Zenodo was built at the European Laboratory for Particle Physics' (CERN) data center to facilitate the sharing and preservation of the long tail of research across all disciplines and scientific domains. Given Zenodo’s long tradition of making research artifacts FAIR (Findable, Accessible, Interoperable, and Reusable), there are still challenges in applying these principles effectively when serving the needs of specific research domains. Plazi’s biodiversity taxonomic literature processing pipeline liberates data from publications, making it FAIR via extensive metadata, the minting of a DataCite Digital Object Identifier (DOI), a licence and both human- and machine-readable output provided by Zenodo, and accessible via the Biodiversity Literature Repository community at Zenodo. The deposits (e.g., taxonomic treatments, figures) are an example of how local networks of information can be formally linked to explicit resources in a broader context of other platforms like GBIF (Global Biodiversity Information Facility). In the context of biodiversity taxonomic literature data workflows, a general-purpose repository’s traditional submission approach is not enough to preserve rich metadata and to capture highly interlinked objects, such as taxonomic treatments and digital specimens. As a prerequisite to serve these use cases and ensure that the artifacts remain FAIR, Zenodo introduced the concept of custom metadata, which allows enhancing submissions such as figures or taxonomic treatments (see as an example the treatment of Eurygyrus peloponnesius) with custom keywords, based on terms from common biodiversity vocabularies like Darwin Core and Audubon Core and with an explicit link to the respective vocabulary term. The aforementioned pipelines and features are designed to be served first and foremost using public Representational State Transfer Application Programming Interfaces (REST APIs) and open web technologies like webhooks. This approach allows researchers and platforms to integrate existing and new automated workflows into Zenodo and thus empowers research communities to create self-sustained cross-platform ecosystems. The BiCIKL project (Biodiversity Community Integrated Knowledge Library) exemplifies how repositories and tools can become building blocks for broader adoption of the FAIR principles. Starting with the above literature processing pipeline, the concepts of and resulting FAIR data, with a focus on the custom metadata used to enhance the deposits, will be explained.

scholarly journals Structure and Function of Multimeric G-Quadruplexes

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3074 ◽  
Author(s):  
Sofia Kolesnikova ◽  
Edward A. Curtis
Keyword(s):  
Nucleic Acid ◽  
Building Blocks ◽  
Structure And Function ◽  
Limited Information ◽  
Functional Elements ◽  
Future Studies ◽  
And Function ◽  
Nucleic Acid Structures ◽  
Sequence Requirements ◽  
Order Structures

G-quadruplexes are noncanonical nucleic acid structures formed from stacked guanine tetrads. They are frequently used as building blocks and functional elements in fields such as synthetic biology and also thought to play widespread biological roles. G-quadruplexes are often studied as monomers, but can also form a variety of higher-order structures. This increases the structural and functional diversity of G-quadruplexes, and recent evidence suggests that it could also be biologically important. In this review, we describe the types of multimeric topologies adopted by G-quadruplexes and highlight what is known about their sequence requirements. We also summarize the limited information available about potential biological roles of multimeric G-quadruplexes and suggest new approaches that could facilitate future studies of these structures.

scholarly journals The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae

Metabolites ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 49 ◽  
Author(s):  
Ana Molina-Márquez ◽  
Marta Vila ◽  
Javier Vigara ◽  
Ana Borrero ◽  
Rosa León
Keyword(s):  
Genetic Transformation ◽  
Selectable Marker ◽  
Genetic Manipulation ◽  
Plant Cells ◽  
Phytoene Desaturase ◽  
Carotenoid Content ◽  
Great Promise ◽  
Encoding Gene ◽  
Average Transformation ◽  
Selection Of

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

scholarly journals Challenges and Advances in Genome Editing Technologies in Streptomyces

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 734 ◽  
Author(s):  
Yawei Zhao ◽  
Guoquan Li ◽  
Yunliang Chen ◽  
Yinhua Lu
Keyword(s):  
Natural Product ◽  
Genome Editing ◽  
Genetic Manipulation ◽  
Chemical Compounds ◽  
Gene Clusters ◽  
Future Research ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Research Focus ◽  
Experimental Conditions

The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.

The Use of Data Explorer as a 3-D Reconstruction Tool for Microscopy Data Sets

1997 ◽  
Vol 3 (S2) ◽  
pp. 1131-1132
Author(s):  
Jansma P.L ◽  
M.A. Landis ◽  
L.C. Hansen ◽  
N.C. Merchant ◽  
N.J. Vickers ◽  
...  
Keyword(s):  
Volume Rendering ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Building Blocks ◽  
General Purpose ◽  
Data Sets ◽  
Laser Scanning Confocal Microscope ◽  
Use Of Data ◽  
Using Data ◽  
Microscopy Data

We are using Data Explorer (DX), a general-purpose, interactive visualization program developed by IBM, to perform three-dimensional reconstructions of neural structures from microscopic or optical sections. We use the program on a Silicon Graphics workstation; it also can run on Sun, IBM RS/6000, and Hewlett Packard workstations. DX comprises modular building blocks that the user assembles into data-flow networks for specific uses. Many modules come with the program, but others, written by users (including ourselves), are continually being added and are available at the DX ftp site, http://www.tc.cornell.edu/DXhttp://www.nice.org.uk/page.aspx?o=43210.Initally, our efforts were aimed at developing methods for isosurface- and volume-rendering of structures visible in three-dimensional stacks of optical sections of insect brains gathered on our Bio-Rad MRC-600 laser scanning confocal microscope. We also wanted to be able to merge two 3-D data sets (collected on two different photomultiplier channels) and to display them at various angles of view.

31 EFFICIENT GENERATION OF klotho MUTATIONS IN PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS USING A DELIVERY OF Cas9 RIBONUCLEOPROTEINS

2017 ◽  
Vol 29 (1) ◽  
pp. 123
Author(s):  
S. Lee ◽  
M. H. Jung ◽  
H. J. Oh ◽  
O.-J. Koo ◽  
B. C. Lee
Keyword(s):  
Somatic Cell ◽  
Genome Editing ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Premature Aging ◽  
Human Diseases ◽  
Limited Information ◽  
Transcription Activator ◽  
Klotho Gene ◽  
Donor Cells

Pigs are useful models for studying human diseases because of the similarity of their anatomy and physiology. Recent advances in genome editing techniques such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat-associated Cas9 system (CRISPR/Cas9) have made it possible to produce animals for specific purposes. Especially, recent application of the CRISPR/Cas9 system improved the efficiency of genome editing in pigs with higher targeting efficiency or percentage of desired mutation compared to other meganucleases (ZFNs and TALENs). The klotho deficiency in small animals such as mice is characterised by an extremely shortened life span with multiple aging-like phenotypes similar to human premature-aging syndromes. However, limited information is available on the function of klotho in large animals such as pigs. The objective of this study was to determine whether the use of non-selected porcine fibroblasts electroporated with Cas9/sgRNA ribonucleoproteins, targeting the klotho gene, for somatic cell nuclear transfer (SCNT) results in high mutation rates in embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and type 2 Cas9 RNPs (total 36 μg, 1:4 ratio, respectively) were transfected into porcine fibroblasts via Neon (Life Technologies) with a single DC pulse of 1400 V for 30 ms. Then, transfected fibroblasts were cultured for 1 day and used randomly for SCNT without selection. SCNT was performed by enucleation of in vitro-matured porcine oocyte, followed by injection of non-selected donor cells, fusion with a single DC pulse of 200 V/mm for 30 μs using an electro cell fusion generator (LF101; Nepa Gene Co.), and electrical activation with a single DC pulse of 150 V/mm for 60 μs using a BTX Electro-Cell Manipulator 2001 (BTX Inc.). The SCNT embryos were cultured in PZM5 culture medium to Day 7 and analysed for the presence of modifications to the klotho gene. Blastocysts were classified as modified if they contained an INDEL as measured by both T7E1 assay and deep sequencing of PCR amplicons spanning the targeted exon. The klotho modification rate was 65% (n = 13), of which 38.5% (n = 5) of the embryos contained biallelic modifications. In conclusion, SCNT with non-selected donor cells transfected with Cas9/sgRNA RNPs might be an efficient and simple tool to produce klotho deficient pigs as models for human diseases. Further studies are required to generate klotho deficient pigs by performing embryo transfer to the recipients. This study was supported by Korea Institute of Planning and Evaluation for Technology in food, agriculture, forestry and fisheries (#311011–05–5-SB010, #114059–03–2-SB010), Research Institute for Veterinary Science, TS Corporation and the BK21 plus program.

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