Abstract
The oleaginous yeast Yarrowia lipolytica has emerged as a powerful alternative for biolipid production due to its high capacity for lipid accumulation. Genetic engineering and synthetic biology are promoted forward to improve production and reroute metabolism for high-value compound synthesis. In this context, efficient, modular, and high-throughput compatible cloning and expression system are required to speed up and rationalize research in this field. Here, we present the fast and modular Golden Gate cloning strategy for the construction of multigene expression vectors and their transformation into Y. lipolytica. As an example, we used the heterologous expression of the carotenoid pathway by cloning three genes involved in this pathway in only one vector allowing reaching production of β-carotene after a single transformation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Beopoulos A, Cescut J, Haddouche R, Uribelarrea J-L, Molina-Jouve C, Nicaud J-M (2009) Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res 48:375–387
Ledesma Amaro R, Nicaud J-M (2016) Metabolic engineering for expanding the substrate range of Yarrowia lipolytica. Trends Biotechnol 34:798–809
Lazar Z, Liu N, Stephanopoulos G (2018) Holistic approaches in lipid production by Yarrowia lipolytica. Trends Biotechnol 36:1157–1170
Darvishi F, Ariana M, Marella ER, Borodina I (2018) Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals. Appl Microbiol Biotechnol 102:5925–5938
Madzak C (2018) Engineering Yarrowia lipolytica for use in biotechnological applications: a review of major achievements and recent innovations. Mol Biotechnol 60:621–635
Larroude M, Rossignol T, Nicaud JM, Ledesma-Amaro R (2018) Synthetic biology tools for engineering Yarrowia lipolytica. Biotechnol Adv 36:2150–2164
Groenewald M, Boekhout T, Neuvéglise C, Gaillardin C, van Dijck PWM, Wyss M. 2013. Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential. Crit Rev Microbiol 0:1–20
Nicaud JM (2012) Yarrowia lipolytica. Yeast 29:409–418
Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden Gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553
Celinska E, Ledesma-Amaro R, Larroude M, Rossignol T, Pauthenier C, Nicaud JM (2017) Golden Gate assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica. Microb Biotechnol 10:450–455
Larroude M, Park Y-K, Soudier P, Kubiak M, Nicaud J-M, Rossignol T (2019) A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology. Microb Biotechnol 12:1249–1259
Barth G, Gaillardin C (1996) Yarrowia lipolytica. In: Wolf K, Breuning KD, Barth J (eds) Non-conventional yeasts in biotechnology. Springer, Berlin, pp 313–388
Larroude M, Celinska E, Back A, Thomas S, Nicaud JM, Ledesma-Amaro R (2018) A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of beta-carotene. Biotechnol Bioeng 115:464–472
Pignede G, Wang H-J, Fudalej F, Seman M, Gaillardin C, Nicaud J-M (2000) Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl Environ Microbiol 66:3283–3289
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Larroude, M., Nicaud, JM., Rossignol, T. (2022). Golden Gate Multigene Assembly Method for Yarrowia lipolytica. In: Mapelli, V., Bettiga, M. (eds) Yeast Metabolic Engineering. Methods in Molecular Biology, vol 2513. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2399-2_12
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2399-2_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2398-5
Online ISBN: 978-1-0716-2399-2
eBook Packages: Springer Protocols