Browsing by Subject "CRISPR/Cas9"

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Capturing wheat phenotypes at the genome level
(2022) Hussain, Babar; Akpınar, Bala A.; Alaux, Michael; Algharib, Ahmed M.; Sehgal, Deepmala; Ali, Zulfiqar; Aradottir, Gudbjorg I.; Batley, Jacqueline; Bellec, Arnaud; Bentley, Alison R.; Cagirici, Halise B.; Cattivelli, Luigi; Choulet, Fred; Cockram, James; Desiderio, Francesca; Devaux, Pierre; Dogramaci, Munevver; Dorado, Gabriel; Dreisigacker, Susanne; Edwards, David; El-Hassouni, Khaoula; Eversole, Kellye; Fahima, Tzion; Figueroa, Melania; Gálvez, Sergio; Gill, Kulvinder S.; Govta, Liubov; Gul, Alvina; Hensel, Goetz; Hernandez, Pilar; Crespo-Herrera, Leonardo Abdiel; Ibrahim, Amir; Kilian, Benjamin; Korzun, Viktor; Krugman, Tamar; Li, Yinghui; Liu, Shuyu; Mahmoud, Amer F.; Morgounov, Alexey; Muslu, Tugdem; Naseer, Faiza; Ordon, Frank; Paux, Etienne; Perovic, Dragan; Reddy, Gadi V. P.; Reif, Jochen Christoph; Reynolds, Matthew; Roychowdhury, Rajib; Rudd, Jackie; Sen, Taner Z.; Sukumaran, Sivakumar; Ozdemir, Bahar Sogutmaz; Tiwari, Vijay Kumar; Ullah, Naimat; Unver, Turgay; Yazar, Selami; Appels, Rudi; Budak, Hikmet
Recent technological advances in next-generation sequencing (NGS) technologies have dramatically reduced the cost of DNA sequencing, allowing species with large and complex genomes to be sequenced. Although bread wheat (Triticum aestivum L.) is one of the world’s most important food crops, efficient exploitation of molecular marker-assisted breeding approaches has lagged behind that achieved in other crop species, due to its large polyploid genome. However, an international public–private effort spanning 9 years reported over 65% draft genome of bread wheat in 2014, and finally, after more than a decade culminated in the release of a gold-standard, fully annotated reference wheat-genome assembly in 2018. Shortly thereafter, in 2020, the genome of assemblies of additional 15 global wheat accessions was released. As a result, wheat has now entered into the pan-genomic era, where basic resources can be efficiently exploited. Wheat genotyping with a few hundred markers has been replaced by genotyping arrays, capable of characterizing hundreds of wheat lines, using thousands of markers, providing fast, relatively inexpensive, and reliable data for exploitation in wheat breeding. These advances have opened up new opportunities for marker-assisted selection (MAS) and genomic selection (GS) in wheat. Herein, we review the advances and perspectives in wheat genetics and genomics, with a focus on key traits, including grain yield, yield-related traits, end-use quality, and resistance to biotic and abiotic stresses. We also focus on reported candidate genes cloned and linked to traits of interest. Furthermore, we report on the improvement in the aforementioned quantitative traits, through the use of (i) clustered regularly interspaced short-palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene-editing and (ii) positional cloning methods, and of genomic selection. Finally, we examine the utilization of genomics for the next-generation wheat breeding, providing a practical example of using in silico bioinformatics tools that are based on the wheat reference-genome sequence.
Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine
(2021) Swietalski, Paul; Hetzel, Frank; Klaiber, Iris; Pross, Eva; Seitl, Ines; Fischer, Lutz
Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine‐5′‐phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed‐batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.
Toward food-grade production of the Bacteroides helcogenes protein-glutamine glutaminase with an optimized Bacillus subtilis strain
(2026) Senger, Jana; Keutgen, Mario; Roth, Nicole; Seitl, Ines; Fischer, Lutz; Senger, Jana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Keutgen, Mario; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Roth, Nicole; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Seitl, Ines; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Fischer, Lutz; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany
Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/Lculture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/Lculture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/Lculture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics.Key points• Site-specific PGB integration enabled by genome sequencing of B. subtilis 007.• Antibiotic-free and secretory PGB production with an optimized B. subtilis host.• Increased PGB production reaching 9.5 µkat/Lculture.