Ultrahigh Throughput Enzyme Screening System Based On Droplet Microreactors

Enzymes are highly efficient,highly selective,and environmentally friendly biocatalysts.Enzymes are playing more and more important roles in chemistry,pharmaceutics,energy,food,environmental protection and other fields.The rapid development of enzyme engineering in industrial application requires more and more novel enzymes and diversed enzymatic functions.Mining new enzyme genes from metagenomics and using molecular directed evolution to improve the properties of existing enzymes are crucial techniques to obtain novel enzyme products.High throughput screening methods are key techniques for searching rare positive genes from huge metagenomics libraries and enzyme mutation libraries,and thus draw worldwild attentions.The ultrahigh throughput screening system based on single-cell microreactor is the newly developed technique.Single-cell microreactor employs pico-liter droplet as the single-cell enzymatic reactor,which can be further analyzed and sorted by a flow cytometer or a microfluidic device,with the throughput of as high as 10~8/day.The microreactor system reduces the volume of each reaction by more than a million times compared with the conventional micro-titer plates,thus greatly decreased the cost of screening reagent.However,there are still a series of drawbacks with single-cell microreactor enzyme screening techniques,such as:(1)the uniformity of the microreactor is poor,which decreases the quantitafication of enzymatic activity as well as the screening efficiency;(2)the flow cytometer for the detection and sorting of microreactors is expensive,limiting the widespread use of the system;(3)due to less screening substrates,only a few enzymes can be applied on this system.To solve the above problems,this thesis carried out the following work:(1)Preparation of high uniformity single cell"water-oil-water"microreactor:we developed a new"water-oil-water"double emulsion droplets preparation method based on membrane-extrusion.The droplets generated by membrane-extrusion were significantly increased in uniformity,thereby greatly enhancing the accuracy of enzymatic activity quantitatification.This sytem can distinguish enzymatic activities within 2-fold.Appling this system,we successfully performed directed evolution towards a thermophilic esterase AFEST.By screening a random mutation library with the volume of two million for only one round of screening,positive mutants with 2-fold increased activities were identifed,and their catalysis efficiencies were close to the limit of molecular diffusion efficiency.Mutants maintained excellent thermal stability and broadened substrate spectrum of the wild-type AFEST,showing 3-fold increased activity towards 7-aminocephalosporanic acid hydrolysis(7-ACA).(2)Microfluidic"water-oil"microreactor based double color fluorescence sorting:microfluidic chips are smart,low cost and flexible,which can integrate droplet generation,manipulation,and analysis modules into a tiny platform.The microfluidic technique has brought new opportunities to the microreactor based high throughput screening.Here we developed a microfluidic"water-in-oil"microreactor based double color fluorescence detection and sorting system.This system can measure two fluorescence signals simultaneously,with throughput of up to 1400/s.Appling this system,we have successfully evolved the enantioselectivity of AFEST through directed evolution.The positive mutants showed both improved enantioselectivity and increased activity towards(S)-ibuprofen,an important anti-inflammatory drug.(3)"Fluorescence droplet entrapment"substrate design strategy:To improve the versatility of single-cell microreactor screening system,we present"fluorescence droplet entrapment,FDE"for designing substrates suitable for microreactors.To verify this stratage,substrates for phosphotriesterase,carboxylesterase,glucosidase,galactosidase system were tested.FDE substrates can strictly control the onset of enzymatic reaction within the droplets and reduce background fluorescence,which greatly improve the accuracy and sensitivity of screening system.We also discussed FDE applications in other important enzyme systems,with the purpose of expanding the versatility of single-cell microreactor ultrahigh screening system.