We broadened the usage of DNA transposon technology by demonstrating its capacity for the rapid creation of expression libraries for long biochemical pathways, which is beyond the classical application of building genome-scale knockout libraries in yeasts. This strategy efficiently leverages the readily available fine-tuning impact provided by the diverse transcriptional environment surrounding each random integration locus. We benchmark the transposon-mediated integration against the nonhomologous end joining-mediated strategy. The latter strategy was demonstrated for achieving pathway random integration in other yeasts but is associated with a high false-positive rate in the absence of a high-throughput screening method. Our key innovation of a nonreplicable circular DNA platform increased the possibility of identifying top-producing variants to 97%. Compared to the classical DNA transposition protocol, the design of a nonreplicable circular DNA skipped the step of counter-selection for plasmid removal and thus not only reduced the time required for the step of library creation from 10 to 5 d but also efficiently removed the "transposition escapers", which undesirably represented almost 80% of the entire population as false positives. Using one endogenous product (i.e., shikimate) and one heterologous product (i.e., (S)-norcoclaurine) as examples, we presented a streamlined procedure to rapidly identify high-producing variants with titers significantly higher than the reported data in the literature. We selected Schef fersomyces stipitis, a representative nonconventional yeast, as a demo, but the strategy can be generalized to other nonconventional yeasts. This new exploration of transposon technology, therefore, adds a highly versatile tool to accelerate the development of novel species as microbial cell factories for producing value-added chemicals.
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.
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