Fluorescence-activated droplet sorting of PET degrading microorganisms

Qiao, Yuxin; Hu, Ran; Chen, Dongwei; Wang, Li; Wang, Zhiyi; Yu, Hao; Fu, Ye; Li, Chunli; Dong, Zhiyang; Weng, Yunxuan; Du, Wei

doi:10.1016/j.jhazmat.2021.127417

Cited by 39 publications

(28 citation statements)

References 39 publications

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“…Meanwhile, two PETase belonging to the carboxylesterase and dienelactone hydrolase family are also identified from these strains. They exhibited the known highest degrading activity against Bis(2-Hydroxyethyl) terephthalate ( Qiao et al, 2021 ). Imidazolinone compounds are worldwide popular herbicides used to cultivate diverse commercial crops.…”

Section: Applicationsmentioning

confidence: 99%

Emerging microfluidic technologies for microbiome research

Wen

et al. 2022

Front. Microbiol.

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The importance of the microbiome is increasingly prominent. For example, the human microbiome has been proven to be strongly associated with health conditions, while the environmental microbiome is recognized to have a profound influence on agriculture and even the global climate. Furthermore, the microbiome can serve as a fascinating reservoir of genes that encode tremendously valuable compounds for industrial and medical applications. In the past decades, various technologies have been developed to better understand and exploit the microbiome. In particular, microfluidics has demonstrated its strength and prominence in the microbiome research. By taking advantage of microfluidic technologies, inherited shortcomings of traditional methods such as low throughput, labor-consuming, and high-cost are being compensated or bypassed. In this review, we will summarize a broad spectrum of microfluidic technologies that have addressed various needs in the field of microbiome research, as well as the achievements that were enabled by the microfluidics (or technological advances). Finally, how microfluidics overcomes the limitations of conventional methods by technology integration will also be discussed.

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Section: Applicationsmentioning

confidence: 99%

Emerging microfluidic technologies for microbiome research

Wen

et al. 2022

Front. Microbiol.

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“…Moreover, the assay described here could be modified for implementation in ultrahigh-throughput format based on droplet microfluidics, with the advantage of detecting real PET hydrolysis products and avoiding the use of surrogate substrates. [25] To that end, the putative workflow comprised of the encapsulation of a PET nanoparticle with the library clone expressing the candidate hydrolase in water-in-oil (w/o) droplets prior to total [34,35] or partial [36] cell lysis. After sufficient incubation for PET hydrolysis, the reaction mix proposed in this work was added to the droplets by picoinjection [37,38] or droplet fusion, [39] following a short incubation to reveal the presence of hydrolysis products before proceeding to on-chip droplet sorting and recovery of the DNA from the droplets by PCR (Figure S9).…”

Section: Discussionmentioning

confidence: 99%

“…First, a fluorescence-activated droplet sorting approach for the discovery of new PET degrading enzymes has been developed that uses fluorescein dibenzoate as a fluorogenic surrogate substrate for TPA esters. [25] On the other hand, a method based on the conversion of PET hydrolysis products, and thus, real substrate, to fluorescent species, such as 2-hydroxyterephthalate from TPA by iron autoxidation. This method was validated before in 96-well microplate using PET granulates [26] and PET nanoparticles, [27] but it is an endpoint assay that requires excitation at UV wavelengths, which is not a feature commonly found in ultrahigh-throughput screening equipment, such as fluorescence-activated cell sorting (FACS) or microfluidic onchip sorters.…”

Section: Introductionmentioning

confidence: 99%

A Coupled Ketoreductase‐Diaphorase Assay for the Detection of Polyethylene Terephthalate‐Hydrolyzing Activity

et al. 2022

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In the last two decades, several PET‐degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high‐throughput screening protocols for PET‐hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described. It was based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that were able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase‐type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methods.

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“…Thus, nearly all other applications require some means to transduce chemical concentration into a detectable signal. This transduction can be accomplished through the use of fluorescent dyes, or with the use of chemical or enzyme-linked reactions. , However, most of these indirect screening applications are usually limited by the lack of specific fluorogenic probes as well as the cytotoxicity, self-hydrolysis, and leakage caused by the probes. To address these problems, we directly link the natural products’ yield with its related synthesis genes’ transcription.…”

Section: Discussionmentioning

confidence: 99%

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

Zhang

Cao

et al. 2022

ACS Synth. Biol.

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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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Fluorescence-activated droplet sorting of PET degrading microorganisms

Cited by 39 publications

References 39 publications

Emerging microfluidic technologies for microbiome research

Emerging microfluidic technologies for microbiome research

A Coupled Ketoreductase‐Diaphorase Assay for the Detection of Polyethylene Terephthalate‐Hydrolyzing Activity

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

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