Leveraging the Hermes Transposon to Accelerate the Development of Nonconventional Yeast-based Microbial Cell Factories

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.