Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome. The development of molecular toolkits and “integration cassettes” have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 “gene drive” system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome.
Acetyl-triacylglycerols (acetyl-TAG) contain an acetate group in the sn-3 position instead of the long-chain fatty acid present in regular triacylglycerol (TAG). The acetate group confers unique physical properties such as reduced viscosity and a lower freezing point to acetyl-TAG, providing advantages for use as emulsifiers, lubricants, and 'drop-in' biofuels. Previously, the synthesis of acetyl-TAG in the seeds of the oilseed crop camelina (Camelina sativa) was achieved through the heterologous expression of the diacylglycerol acetyltransferase gene EaDAcT, isolated from Euonymus alatus seeds that naturally accumulate high levels of acetyl-TAG. Subsequent work identified a similar acetyltransferase, EfDAcT, in the seeds of Euonymus fortunei, that possesses higher in vitro activity compared to EaDAcT. In this study, the seed-specific expression of EfDAcT in camelina led to a 20 mol% increase in acetyl-TAG levels over that of EaDAcT. Coupling EfDAcT expression with suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation, up to 90 mol% in the best transgenic lines. Accumulation of high levels of acetyl-TAG was stable over multiple generations, with minimal effect on seed size, weight, and fatty acid content. Slight delays in germination were noted in transgenic seeds compared to the wild type. EfDAcT transcript and protein levels were correlated during seed development with a limited window of EfDAcT protein accumulation. In high acetyl-TAG producing lines, EfDAcT protein expression in developing seeds did not reflect the eventual acetyl-TAG levels in mature seeds, suggesting that other factors limit acetyl-TAG accumulation.
Acetyl-triacylglycerols (acetyl-TAG) produced in the seeds of different Euonymus species are triacylglycerols (TAG) that possess an sn-3 acetate group instead of a long chain fatty acid. This unusual structure confers useful properties to acetyl-TAG, including reduced kinematic viscosity and improved cold temperature performance. Acetyl-TAG are synthesized by unique diacylglycerol acetyltransferases (DAcTs), expressed in the endosperm of Euonymus seeds, that use acetyl-CoA to acetylate the sn-3 position of diacylglycerol (DAG) molecules. Isolation and expression of DAcT enzymes from different Euonymus species has resulted in the successful accumulation of high levels of acetyl-TAG in different oil seed crops. For example, expression of EfDAcT isolated from E. fortunei caused acetyl-TAG levels of 81 mol% in camelina seeds and 51 mol% in pennycress. To increase acetyl-CoA supply for EfDAcT, CRISPR-based genome editing was used to generate mutations in FATTY ACID ELONGASE1 (FAE1) genes in both camelina and pennycress. FAE1 is a component of the fatty acid elongase complex that uses acetyl-CoA to produce the very long-chain fatty acids found in the seed oil of Brassicaceae species. Consistent with the hypothesis that eliminating FAE1 function results in more acetyl-CoA availability for acetyl-TAG synthesis, expression of EfDAcT in fae1 mutants resulted in very high levels of acetyl-TAG accumulation. This was particularly evident in pennycress fae1 lines where acetyl-TAG levels exceeded 95 mol% in the best transgenic lines. These results demonstrate the usefulness of genome editing to modify the genetic background of oilseed crops to enhance production of a targeted product.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.