Fluorescence-Activited Droplet Sorting Based Ultrahigh Throughput Screening System For Lipase/Esterase

Lipases and esterases are two kinds of important industrial enzymes which possessvarious applications in industrial fields such as food, fine chemical, detergent,medicine, lipid and energy. However, natural lipase/esterase often need furtherengineering to fit the application requirements due to their unstablity, inactivity tounnatural substrates, poor stereoselectivity. Directed evolution is a powerful enzymeengineering method which has been successfully used in improving catalyticcharacteristics for many enzymes. However, the success of directed evolution dependson screening of large random mutantion libraries and is challenging for traditionalscreening methods which are low throughput, laborious and cost consuming.To meet the requirement of high throughput, high versatility screening methods,this thesis explored the application of fluorescence-activited droplet sorting (FADS)system for lipase/esterase ultrahigh throughput screening.The schematic of FADSsystem is to compartmentalize different enzyme mutants along with fluorogenicsubstrate into individual droplets with the diameter of merely~10μm to formdroplet-based microreactors, which can be detected and sorted by flow cytometry at avery high speed according to their fluorescence intensity. This screening methodcombining the merits of high versatility and tiny reaction volumn of droplet-basedmicroreactors and the high sensitivity and high speed of flow cytometry, thus wouldbe one of the promising method for enzyme screening.There are several technical requirements to develop a successful FADS screeningsystem. Firstly, target enzyme molecules should be displayed on the host cell surfaceto contact with the substrate. Secondly, stable water-in-oil-in-water droplet is neededas the carrier of the microreactor, which offers a linkage between genotype andphenotype. Thirdly, the amont of cells incapsulated should be optimized in order to achieve a good single-cell compartmentalization. Last but no least, proper fluorogenicsubstrate with good retention in the microreactior is crucial for the efficiency of thesystem.In this thesis, ice nucleation protein (INP) cell surface display system was appliedto display a thermophilic lipase AFEST from Archaeoglobus fulgidus on the surfaceof E. coli JM109strain. Then a membrane extruding method was developed togenerate water-in-oil-in-water droplets with diameter of~20μm, which were stableduring enzymatic reaction and suitable for FADS. After cell encapsulation efficienceoptimization, we discovered that5×107cells dispersed in1.5mL water-in-oil-in-waterdroplets could obtain satisfying encapsulation efficience. Fluorescein dibutyrate waschosen as the model substrate because of the good retention of the product fluoresceinin the microreactor. At last, model screening experiments were performed by varyingthe positive-negative ratio and screening threshold. The best enrichment factorexceeded1000folds, which indicated the promising screening ability of this FADSsystem.The FADS system developed by this thesis can achieve an excellent throughput ashigh as>107, which is valuable for the study of this important industrial enzymefamily. Besides, FADS system can also be used in the ultrahigh thoughput screeningof other hydrolases as well as oxydoreductases. Thus our research on some key stepsin FADS system, such as droplet generation, enzymatic reaction inside microreactors,and detecting and sorting of microreactors by flow cytometry, has general meaning forscreening of these kinds of enzymes.