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Abstract:
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 dassical 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 S 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 Scheffersomyces 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.
