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.
Similar content being viewed by others
REFERENCES
Madzak, C., Gaillardin, C., and Beckerich, J.M., Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review, J. Biotechnol., 2004, vol. 109, nos. 1–2, pp. 63–81. https://doi.org/10.1016/j.jbiotec.2003.10.027
Madzak, C., Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering, Appl. Microbiol. Biotechchnol., 2015, vol. 99, no. 11, pp. 4559–4577. https://doi.org/10.1007/s00253-015-6624-z
Xue, Z., Sharpe, P.L., Hong, S.P., et al., Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica,Nat. Biotechnol., 2013, vol. 31, no. 8, pp. 734–740. https://doi.org/10.1038/nbt.2622
Groenewald, M., Boekhout, T., Neuvéglise, C., et al., Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential, Crit. Rev. Microbiol., 2014, vol. 40, no. 3, pp. 187–206. https://doi.org/10.3109/1040841X.2013.770386
Fickers, P., Le Dall, M.T., Gaillardin, C., et al., New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica,J. Microbiol. Methods, 2003, vol. 55, no. 3, pp. 727–737. https://doi.org/10.1016/j.mimet.2003.07.003
Verbeke, J., Beopoulos, A., and Nicaud, J.M., Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains, Biotechnol. Lett., 2013, vol. 35, no. 4, pp. 571–576. https://doi.org/10.1007/s10529-012-1107-0
Liu, L. and Fan, X.-D., CRISPR–Cas system: a powerful tool for genome engineering, Plant Mol. Biol., 2014, vol. 85, pp. 209–218. https://doi.org/10.1007/s11103-014-0188-7
Barrangou, R., Fremaux, C., Deveau, H., et al., CRISPR provides acquired resistance against viruses in prokaryotes, Science, 2007, vol. 315, no. 5819, pp. 1709–1712. https://doi.org/10.1126/science.1138140
Wiedenheft, B., Sternberg, S.H., and Doudna, J.A., RNA-guided genetic silencing systems in bacteria and archaea, Nature, 2012, vol. 482, no. 7385, pp. 331–338. https://doi.org/10.1038/nature10886
Khanzadi, M.N. and Khan, A.A., CRISPR/Cas9: nature’s gift to prokaryotes and an auspicious tool in genome editing, J. Basic Microbiol., 2019, pp. 1–12. https://doi.org/10.1002/jobm.201900420
Raschmanová, H., Weninger, A., Glieder, A., et al., Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: current state and future prospects, Biotechnol. Adv., 2018, vol. 36, no. 3, pp. 641–665. https://doi.org/10.1016/j.biotechadv.2018.01.006
Jinek, M., Chylinski, K., Fonfara, I., et al., A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity, Science, 2012, vol. 337, no. 6096, pp. 816–821. https://doi.org/10.1126/science.1225829
Schwartz, C., Shabbir-Hussain, M., Frogue, K., et al., Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica,ACS Synth. Biol., 2016, vol. 6, no. 3, pp. 402–409. https://doi.org/10.1021/acssynbio.6b00285
Beopoulos, A., Nicaud, J.M., and Gaillardin, C., An overview of lipid metabolism in yeasts and its impact on biotechnological processes, Appl. Microbiol. Biotechnol., 2011, vol. 90, no. 4, pp. 1193–1206. https://doi.org/10.1007/s00253-011-3212-8
Yuzbasheva, E.Y., Agrimi, G., Yuzbashev, T.V., et al., The mitochondrial citrate carrier in Yarrowia lipolytica: its identification, characterization and functional significance for the production of citric acid, Metab. Eng., 2019, vol. 54, pp. 264–274. https://doi.org/10.1016/j.ymben.2019.05.002
Boulton, C.A. and Ratledge, C., Correlation of lipid accumulation in yeasts with possession of ATP: citrate lyase. Microbiology, 1981, vol. 127, no. 1, pp. 169–176. https://doi.org/10.1099/00221287-127-1-169
Dulermo, T., Lazar, Z., Dulermo, R., et al., Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis, Biochim. Biophys. Acta, 2015, vol. 1851, no. 9, pp. 1107–1117. https://doi.org/10.1016/j.bbalip.2015.04.007
Sambrook, J., Maniatis, T., and Fritsch, E., Molecular Cloning: A Laboratory Mannual, 2nd ed., New York, USA: Cold Spring Harbor Laboratory Press, 1989.
Gibson, D.G., Young, L., Chuang, R.Y., et al., Enzymatic assembly of DNA molecules up to several hundred kilobases, Nat. Methods, 2009, vol. 6, no. 5, pp. 343–345. https://doi.org/10.1038/nmeth.1318
Schwartz, C.M., Hussain, M.S., Blenner, M., and Wheeldon, I., Synthetic RNA polymerase III promoters facilitate high-efficiency CRISPR-Cas9-mediated genome editing in Yarrowia lipolytica,ACS Synth. Biol., 2016, vol. 5, no. 4, pp. 356–359. https://doi.org/10.1021/acssynbio.5b00162
Blazeck, J., Liu, L., Redden, H., and Alper, H., Tuning gene expression in Yarrowia lipolytica using a hybrid promoter approach, Appl. Environ. Microbiol., 2011, vol. 77, no. 22, pp. 7905–7914. https://doi.org/10.1128/AEM.05763-11
Yuzbasheva, E.Y., Mostova, E.B., Andreeva, N.I., et al., Co-expression of glucose-6-phosphate dehydrogenase and acyl-CoA binding protein enhances lipid accumulation in the yeast Yarrowia lipolytica,N. Biotechnol., 2017, vol. 39, pp. 18–21. https://doi.org/10.1016/j.nbt.2017.05.008
Labun, K., Montague, T.G., Gagnon, J.A., et al., CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering, Nucleic Acids Res., 2016, vol. 44, no. W1, pp. 272–276. https://doi.org/10.1093/nar/gkw398
Yuzbasheva E.Yu., Yuzbashev T.V., Konstantinova T.K. et al. The ability of the N- and C-domains of the cell wall protein homologue of Saccharomyces cerevisiae Flo1p to expose Lip2 lipase on the surface of Yarrowia lipolytica yeast cells, Biotekhnologiya, 2011, no. 1, pp. 23–33. https://doi.org/10.1134/S000368381108011
van Rossum, H.M., Kozak, B.U., Pronk, J.T., and van Maris, A.J., Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: pathway stoichiometry, free-energy conservation and redox-cofactor balancing, Metab. Eng., 2016, vol. 36, pp. 99–115. https://doi.org/10.1016/j.ymben.2016.03.006
Swiegers, J.H., Dippenaar, N., Pretorius, I.S., and Bauer, F.F., Carnitine-dependent metabolic activities in Saccharomyces cerevisiae: three carnitine acetyltransferases are essential in a carnitine-dependent strain, Yeast, 2001, vol. 18, no. 7, pp. 585–595. https://doi.org/10.1002/yea.712
Strijbis, K., van Roermund, C.W., van den Burg, J., et al., Contributions of carnitine acetyltransferases to intracellular acetyl unit transport in Candida albicans,J. Biol. Chem., 2010, vol. 285, no. 32, pp. 24335–24346. https://doi.org/10.1074/jbc.M109.094250
Chen, Y., Zhang, Y., Siewers, V., and Nielsen, J., Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase, FEMS Yeast Res., 2015, vol. 15, no. 3, p. fov015. https://doi.org/10.1093/femsyr/fov015
Otto, C., Yovkova, V., Aurich, A., et al., Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica,Appl. Microbiol. Biotechnol., 2012, vol. 95, no. 4, pp. 905–917. https://doi.org/10.1007/s00253-012-4085-1
ACKNOWLEDGMENTS
The work was carried out using the equipment of the Unique Scientific Facility of the All-Russia Collection of Industrial Microorganisms National Bioresource Center of the Kurchatov Institute National Resource Center (GOSNIIgenetika).
Funding
The work was financially supported by the Russian Federation (State Task no. 595-00003-19 PR) and partially funded by grant no. MK-2241.2019.7.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
The authors declare that they have no conflicts of interest.
This article does not contain any studies involving animals performed by any of the authors.
This article does not contain any studies involving human participants performed by any of the authors.
Additional information
Translated by I. Gordon
Abbreviations: 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.
Rights and permissions
About this article
Cite this article
Yuzbasheva, E.Y., Yuzbashev, T.V., Vinogradova, E.B. et al. Inactivation of Yarrowia lipolytica YlACL2 gene Coding Subunit of ATP Citrate Lyase Using CRISPR/Cas9 System. Appl Biochem Microbiol 56, 885–892 (2020). https://doi.org/10.1134/S0003683820090112
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0003683820090112