Inactivation of Yarrowia lipolytica YlACL2 gene Coding Subunit of ATP Citrate Lyase Using CRISPR/Cas9 System
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UCERS, BIOLOGY, SELECTION, AND GENE ENGINEERING
Inactivation of Yarrowia lipolytica YlACL2 gene Coding Subunit of ATP Citrate Lyase Using CRISPR/Cas9 System E. Y. Yuzbashevaa, *, T. V. Yuzbashevb, E. B. Vinogradovaa, I. M. Kosikhinaa, M. O. Taratynovaa, **, D. A. Dementeva, A. I. Solovyevc, D. A. Egorovac, and S. P. Sineokya aRussian
State Collection of Industrial Microorganisms National Bioresource Center (BRC VKPM), State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIgenetika), Moscow, 117545 Russia bDepartment of Bioengineering, Imperial College London, London SW7 2AZ UK c Federal National Research Center of Epidemiology and Microbiology named N.F. Gamaleya, Russian Ministry of Health, Moscow, 123098 Russia *e-mail: [email protected] **e-mail: [email protected] Received November 21, 2019; revised January 14, 2020; accepted January 22, 2020
Abstract—In this study, YlACL2 was inactivated by two methods: traditional approach based on homologous recombination and uracil marker and markerless system using CRISPR/Cas9. The efficiency of YlACL2 inactivation using traditional approach was 4% (one ΔYlacl2 strain out of 24 tested transformants) whereas knockout efficiency using CRISPR/Cas9 system was 75% (18 ΔYlacl2 strains out of 24 tested transformants). YlACL2 null mutant strains were not able to utilize citrate as a single carbon source. Growth kinetics was investigated in the media with glucose and acetate as a single carbon source. The fact that ΔYlacl2 is able to grow in the minimal medium with glucose as a single carbon source provides evidence that there is an alternative source of acetyl-CoA on carbohydrate substrates in Y. lipolytica. Keywords: Yarrowia lipolytica, CRISPR/Cas9 system, ATP citrate lyase, YlACL2 DOI: 10.1134/S0003683820090112
INTRODUCTION Yarrowia lipolytica is obligatory aerobic ascomycetous heterothallic dimorphic yeast. It can use glucose, glycerol, alcohols, acetate, citrate, and hydrophobic substrates (such as alkanes, fatty acids, and triacylglycerides) for its growth [1]. The industrial use of Y. lipolytica yeast as a feed protein source was started in 1950 at British Petroleum (Great Britain) [2]. The intensive use of genetic engineering and metabolic engineering methods for the development of Y. lipolytica industrial strains began in 2000; furthermore, a number of preparations based on strains of this species have been launched on the market. For example, DuPont (United States) has designed preparations enriched with omega-3 fatty acids for use as a fish feed additive (VerlassoTM) and a food supplement (New HarvestTM) [2, 3]. The Y. lipolytica yeast has established itself as a microorganism that is suitable for the construction of genetically modified strains, and nowAbbreviations: ACL—ATP citrate lyase; CRISPR—clustered regularly interspaced short palindromic repeats; OD600 —optical density at a wavelength of 600 nm; PAM—protospacer adjacent motif; sgRNA—small guide RNA.
adays, the so-called GILSP (Good
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