Ultrahigh throughput screening for enzyme function in droplets

Recent trends in biocatalysis

“…Importantly, if an aptamer for one enantiomer is available, the other enantiomer is easily detectable by synthesising the opposite enantiomer of the aptamer. 50 This figure was inspired by Kintses et al and Neun et al 52,53 sort droplets into multiple populations. 38 Equally important is the ability to sort droplets based on more than one fluorescence measurement, which facilitates for example the evolution of enantioselective enzymes (Fig.…”

“…While droplets have been used to screen hydrolase, oxidoreductase, aldolase, transferase, and isomerase activities, the hydrolases dominate by far (lipase, esterase, phosphatase, phosphonate hydrolase, sulfatase, b-glucosidase, b-galactosidase, and more). 42,[53][54][55][56] The reason for this is simply that most droplet sorting systems require a fluorescent signal and that it is relatively simple to design and synthesise fluorogenic hydrolase substrates. Four years ago, the Hollfelder group broadened the applicability of droplet sorting by introducing absorbanceactivated droplet sorting (AADS) (Fig.…”

“… 65 Mass spectrometry (MS) has been used to detect droplet contents at rates up to 30 Hz, 66 and sorting was reported at rates of about 6 Hz, using electrospray ionization (ESI)-MS to analyse ∼15 000 droplets in 6 h. 46 While droplets containing an in vitro expressed transaminase could be sorted, the large droplet volume (25 nanolitre compared to 2 picolitre for fluorescence activated droplet sorting (FADS)) means that this detection strategy would not be applicable to biocatalysts derived from single DNA molecules or single cells. 53 However, recent advances in in vitro DNA amplification, transcription, translation, and assay in microfluidic droplets could address this issue. 67 Another limitation of MS detection is that it is destructive, so droplets have to be split before analysis.…”

“…An excellent summary by Neun et al shows that despite all the progress, only fluorescence, absorbance, and electrochemical detection has been used for the sorting of actual directed evolution-derived or metagenome libraries. 53 Furthermore, fluorescence detection is still unique in being the only format that uses droplets of only a few picolitres (high enzyme concentration) and is capable of detecting low nanomolar product concentrations and sorting at frequencies of several kHz. High-speed absorbance measurements on picolitre and femtolitre droplets is possible using differential detection photothermal interferometry (DDPI).…”

A Titratable Cell Lysis-on-Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution

“… 5 − 7 Here the screening scale is in the picoliter droplet-range with single-cell encapsulation—ensured via Poisson distribution—to maintain a genotype-to-phenotype linkage. 5 , 8 , 9 In functional metagenomics, microbes serve as living cell factories to transform the genetic information encoded by eDNA into functional enzymes for screening. The model organism Escherichia coli is the typical workhorse of choice due to the ease of handling and the availability of a wide range of genetic tools.…”