Development And Application Of A Droplet-Based Microfluidic System For High-Throughput Characterization And Screening Of Streptomyces

As the largest genus of actinomycetes,Streptomyces is widely distributed in nature,and due to the complexity of Streptomyces genome,it can produce a variety of secondary metabolites.Currently,more than half of the antibiotics commonly used are either directly produced from Streptomyces or their derivatives.Streptomyces can also produce a variety of other natural products with high added value.Therefore,the attention and research on Streptomyces is increasing.At present,research on Streptomyces is mainly focused on the genetic engineering and screening,which is no longer a big problem due to the continuous advancement of molecular biology techniques such as gene amplification and genome editing.However,screening throughput becomes the bottleneck once a large number of engineered strains was generated.The screening of Streptomyces is still dependent on low-throughput screening methods such as well 24 or 96-well plates.The screening of the plate can only operate tens to hundreds of strains at a time.Such throughput is far from optimal.In the case of recently reported Streptomyces screening by flow cytometry,it has the disadvantage of not being able to screen extracellular metabolites,high false positive rate and the severe damage to cells.Therefore,an alternative widely applicable high-throughput screening method is in urgent need.The use of droplet microfluidic technology for strain screening is become common and shows strong advantages.But so far,no droplet microfluidic technology has been applied to Streptomyces.Here,we developed a droplet-based microfluidic technology to characterize and screen Streptomyces.Firstly,we optimized droplet generation and incubation method so that streptomyces spore can germinate and grow normally.Then,we constructed plasmids with different promoters in front of egfp gene,and transformed into S.lividans.We obtained strains with different fluorescence strength to validate our method of characterization and screening Streptomyces via droplet microfluidic technology.The single sorting results showed that the enrichment rate increased from about 3.2%before sorting to 91.7%after sorting,which increased by about 29 times,and the throughput reached 36,000 cells/h,which improved the screening efficiency by nearly a thousand times compared with traditional methods.Lastly,we combined the droplet microfluidic technology with biosensor technology to screen high erythromycin producer.