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 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.
Keywords: (S)-norcoclaurine; Hermes transposon; genome integration; nonconventional microbes; nonhomologous end joining; shikimate.