Investigating the role of noncoding regulatory DNA in plasmid
development for Yarrowia lipolytica
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 4.5-fold increase in gene expression and 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.