Transcription-Associated Fluorescence-Activated Droplet Sorting for Di-rhamnolipid Hyperproducers

Abstract

Rhamnolipids (RLs) are biosurfactants with great economic significance that have been used extensively in multiple industries. Pseudomonas aeruginosa is a promising microorganism for sustainable RL production. However, current CTAB-MB based screening of RL-producing strains is time-consuming, labor-intensive, and unable to distinguish monoand di-RL. In this study, we developed a novel transcription-associated fluorescence-activated droplet sorting (FADS) method to specifically target the di-RL hyperproducers. We first investigated critical factors associated with this method, including the specificity and sensitivity for discriminating di-RL overproducers from other communities. Validation of genotype−phenotype linkage between the GFP intensity, rhlC transcription, and di-RL production showed that rhlC transcription is closely correlated with di-RL production, and the GFP intensity is responsive to rhlC transcription, respectively. Using this platform, we screened out ten higher di-RL producing microorganisms, which produced 54−208% more di-RL than the model P. aeruginosa PAO1. In summary, the droplet-based microfluidic platform not only facilitates a more specific, reliable, and rapid screening of P. aeruginosa colonies with desired phenotypes, but also shows that intracellular transcription-associated GFP intensity can be used to measure the yield of di-RL between populations of droplets containing different environmental colonies. This method also can be integrated with transposon mutation libraries to target P. aeruginosa mutants.

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“…The droplets‐based sorting enables cell proliferation inside droplets, achieving a considerable cell density to express desired enzymes, whereas the fluorescence‐activated cell sorting (FACS) technique is not suitable to do this. FADS technology has been successfully used for the directed evolution of enzymes (Qiao et al, 2018 ), single‐cell analysis (Vallejo et al, 2019 ) and screening for improved microbial production of extracellular compounds (Oberpaul et al, 2022 ; Xu et al, 2022 ). For most of the FADS screening, it is essential to convert the desired phenotype into a detectable signal.…”