Yuxin Zhao

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.

This work reports a paper-based sensor technology for point-of-care nucleic acid amplification tests. The technology integrates a sensor chip and an Arduino-compatible fluorescence detector. The paper-based sensor provides the functions of reagent storage, sample transportation, and DNA amplification. The compact and low-cost fluorescence detector can quantify the amplicons in real time. The results show that the paper-based sensor can detect genomic DNA extracted from Escherichia coli with the concentration as low as 2 × 10 ⁴ copies/ µL. As a diagnosis tool, the sensor has the potential to measure multiple target genes simultaneously.

Summary: L ysobacter enzymogenes is an important biocontrol agent with the ability to produce a variety of lytic enzymes and novel antibiotics. Little is known about their regulatory mechanisms. Understanding these will be helpful for improving biocontrol of crop diseases and potential medical application. In the present study, we generated an hfq (encoding a putative ribonucleic acid chaperone) deletion mutant, and then utilized a new genomic marker‐free method to construct an hfq ‐complemented strain. We showed for the first time that H fq played a pleiotropic role in regulating the antibacterial antibiotic biosynthesis and extracellular lytic enzyme activity in L . enzymogenes . Mutation of hfq significantly increased the yield of WAP ‐8294 A 2 (an antibacterial antibiotic) as well as the transcription of its key biosynthetic gene, waps1 . However, inactivation of hfq almost abolished the extracellular chitinase activity and remarkably decreased the activity of both extracellular protease and cellulase in L . enzymogenes . We further showed that the regulation of hfq in extracellular chitinase production was in part through the impairment of the secretion of chitinase A . Collectively, our results reveal the regulatory roles of hfq in antibiotic metabolite and extracellular lytic enzymes in the underexplored genus of L ysobacter .