On-chip Screening Of Mutant Strains And Assay Of Their Metabolites

The increase of antimicrobial resistance and the outbreak of new diseases have brought unprecedented challenges with antibiotic therapy,and discovering and developing effective compounds to fight against infections or diseases have become a challenge.Microbial natural products with a wide range of biological activities caused by structural diversity remain a propitious source of novel antibiotics or active compounds.However,the traditional methods such as synthetic biology,metabolic engineering,microbial genome mining,and biomass extraction still have a long time span,complex screening,and cumbersome operations when screening mutant strains using conventional flasks and agar plates.In recent years,the wide applications of droplet microfluidic techniques in life science provide new powerful tools for microbial analysis and detection.Especially in the single-cell level analysis and detection of bacteria,droplet microfluidic techniques have advantages,such as short reaction time and high analytical sensitivity.In this study,a droplet-based microfluidic platform included a flowfocusing microfluidic device and an integrated microfluidic device combined with HPLCMS/MS was developed for screening chemically induced mutant strains and analyzing their metabolites.Using this platform,microbial mutant strains could be screened out in 4 h.The microfluidic method could efficiently screen microbial mutants with multiple advantages over the traditional method such as accurate control of experimental conditions,less sample consumption,and simplified cumbersome operations.This research provides a new idea for the exploration and research of the new technology and analysis method for rapid screening of microbial mutants,and provides a powerful tool for accelerating the mining of natural products.The results of this research are as follows:1.Eight antibiotics were selected,including gentamicin,streptomycin,kanamycin,rifamycin,penicillin,cephalexin,erythromycin,ofloxacin,and the concentration gradients were 2 ?g/m L,4 ?g/m L,6 ?g/m L,8 ?g/m L,10 ?g/m L,12 ?g/m L and 14 ?g/m L of each antibiotic.Under the gradient screening of eight antibiotics,two strains of antibiotic-sensitive actinomycetes Str7 and Str10 were screened out.To obtain mutant strains of Str7 and Str10,a concentration of 10 ?g/m L gentamicin and 10 ?g/m L kanamycin were used for strain Str7,and a concentration of 10 ?g/m L streptomycin and 10 ?g/m L kanamycin were used for strain Str10.The salt tolerance of strains was tested by using NaCl with a concentration gradient of5 %,10 %,15 %,16 %,17 %,18 %,19 %,20 %,and four strains of high salt-sensitive strain Bac1,Bac2,Bac5,and Bac6 were screened out.To obtain mutant strains with high salt tolerance,the concentration of 18 % NaCl was used for strain Bac1,Bac2,Bac5,and Bac6 as the screening concentration.In order to identify six strains,16 S r DNA sequencing was performed.The results of strain identification were two Streptomyces strains(Str7 and Str10),two Salinicoccus strains(Bac1 and Bac2),a Halomonas strain(Bac5)and a Planomicrobium strain(Bac6).2.Six gradients of mineral oil flow rate(Qo)and aqueous phase flow rate(Qw)ratios were taken into consideration to explore the relationship between the Qo/Qw and droplet diameters.On the basis of the result,the Qo/Qw ratio of 0.50/0.75(the flow rates of mineral oil and aqueous phase at 0.50 ?L/min,0.75 ?L/min,respectively.)was then used to obtain uniform size droplets of 20 ?m in diameter using the flow-focusing microfluidic device.In order to determine the strain concentration for droplet encapsulation,8.0 × 107 CFU/m L,1.6× 108 CFU/m L and 4.0 × 108 CFU/m L were used to optimize the occupancy and strain number distribution in droplets.When encapsulating strains into the droplets,the strain number distribution in the droplets followed Poisson statistics.When strain suspension reached 4 ×108 CFU/m L,the fraction of droplets containing no strains was 5.56%,whereas those containing one strain was 16.06%,and those containing two or more strains was 78.38%.3.An integrated microfluidic device was designed and fabricated in the study,which was used to screen and selectively extract the target droplets containing mutant strains.This device was composed of five layers: a top control layer,a middle control layer,a culture/analysis layer,a supporting layer,and a glass slide.The culture/analysis layer had an array of 144 lecotropal single-droplet capture units(12 × 12;height: 25 ?m;length: 108 ?m;width: 80 ?m)where single droplets could be captured and analyzed.The top and middle control layers of the device had 12-row and 12-column microvalve systems,respectively.When the two control layers were aligned and bonded together,a total of 144 junctions were generated.The 144 junction areas matched the 144 single-droplet trapping units in the culture/analysis layer.Using the two sets of microvalve systems,the device could perform droplet trapping,purging,and extracting processes.In subsequent work,the concentration of strain suspension(4 × 108CFU/m L)and the flow rates 0.75 ?L/min for the dispersed phase and 0.50 ?L/min for the oil phase were used for droplet encapsulation of microbial strains(two actinomycetes and four bacteria).When the hydraulic pressure was only applied to the top or middle control layer under a pressure of 19 psi or 10 psi,the capture efficiency of a single droplet in the device was75.80% ± 3.20%.Using the two sets of microvalve systems,the target droplets containing mutant strains could be screened out in 4 h.4.In this study,combined with HPLC-MS/MS technology,the traditional screening method was simultaneously used as control,and the secondary metabolites of wild-type and screened mutant-type strains of six strains were analyzed respectively.Multivariate and heatmap analysis showed that the characteristic peaks of the mutant strains screened by the microfluidic method and traditional method(agar plates)were clustered and separated evidently from the wild-type strains.The results showed that the metabolic compositions of mutant strains screened by the two methods were consistent and significantly different from that of wild strains.The top 20 differential characteristic peaks of all samples were annotated with the Human Metabolome Database(HMDB),and the significant differences of the characteristic peaks indicated that the mutant strains screened by the microfluidic method and traditional method had highly similar metabolic compositions.All the results verified the reliability of the on-chip screening method and the stability of data obtained from this method.Therefore,the on-chip screening method is effective for screening chemically induced microbial mutants.This method can reduce screening timescales and simplify cumbersome operations compared with the traditional screening method.This microfluidic screening method is suitable not only for actinomycete mutant screening,but also for bacterial mutant screening with wide applicability and reliability.