Abstract
Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.
Key points
• In Y. lipolytica, an ARS comprises an origin of replication, a spacer, and a centromere.
• The unannotated spacer present in the wild-type ARS stabilizes episomal expression.
• Plasmid stability and intramolecular rearrangements exhibit a clear cargo dependency.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
Flow cytometry analysis was performed by Dr. Shawn Rigby from the Office of Biotechnology at Iowa State University.
Funding
This work was supported by the National Science Foundation Grants (1716837 and 1749782) and the Iowa State University bio-based product seed grant. ZS was also partically funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018420).
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C. L., M. C., and Z. S. conceived the idea; C. L. and M. C. performed the initial experiments to evaluate the effect of ARS selection on plasmid behavior; C. L. and Z. Y. constructed the plasmids with the extended foreign spacer N1 to N6; C. L. performed all the rest of the experiments and analyzed the data; C. L. and Z. S. wrote the manuscript. All the authors read and approved the final manuscript.
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Lopez, C., Cao, M., Yao, Z. et al. Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering. Appl Microbiol Biotechnol 105, 5959–5972 (2021). https://doi.org/10.1007/s00253-021-11399-4
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DOI: https://doi.org/10.1007/s00253-021-11399-4