(157ar) Leveraging the Hermes Transposon to Accelerate the Development of Nonconventional Yeast-Based Microbial Cell Factories

Authors: 
Zhao, Y. - Presenter, Iowa State University
Ploessl, D., Iowa State University
Ghosh, S., Iowa State University
Gao, M., Iowa State University
Cao, M., University of Illinois at Urbana-Champaign
Shao, Z., Iowa State University
Yao, Z., Iowa State Univeristy
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 provided by the diverse transcriptional environment surrounding each random integration locus. We benchmark the transposon-mediated integration against the non-homologous end joining-mediated strategy. The latter strategy has been 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 not only 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 days 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.