Construction and Application of High-throughput Screening Platforms for Development of Industrial Microorganism Based on Microplate Technology and Microfluidic Technology
Author:Zhu Xu Dong
Supervisor:chu ju wang yong hong
High throughput screening is based on high throughput devices and techniques that automate the processing of tiny volumes of liquid simultaneously with advanced signal detection and accurate data analysis to screen out target compounds,genes,enzymes from living cells or strains.The development trend of high-throughput screening devices is miniaturization,automation,standardization and robotics.For industrial microbial optimization,high-throughput screening is the key technology.Our subject focuses on the live cell screening technology,high-throughput screening technology based on microplate and the ultra-high throughput screening technology based on microfluidic chip were explored in this study.Firstly,a complete high-throughput screening process including cultivation and screening based on microplate was developed to acquire improved Aspergillus niger with elevated production of glucoamylase.The cultivation of Aspergillus niger was achieved with well-dispersed morphology in 48-deep-well micro-titer plate.Further,we discovered the close negative correlation between glucoamylase and pH of the fermentation broth.A novel high-throughput analysis method,using methyl-orange by spectro-photometric,was constructed.When compared with the conventional analysis method using 4-4-nitrophenol-a-D-glucopyranoside as substrate,they showed a close agreement,whose correlation coefficient was 0.96 by statistical analysis.Utilizing this novel high-throughput screening methods,we finally acquired a strain with activity of 2229 AGI/mL,69.9%higher yield of glucoamylase than parent strains.Secondly,we established a dual-index high-throughput assay that was able to simultaneously analyze high-yield extracellular enzymes and enzyme secretion ratios of glucose oxidase(GOD)from Aspergillus niger.A method of enzyme activity determination suited for 96-well plates was constructed,so that the time of enzyme analysis was reduced from 5 min/sample to 0.5 min/sample.At the same time,the relationship between GOD activity and substrate carbon source metabolism was found.Based on this,a rapid analysis method based on residual sugar analysis was established.Compared with the traditional method relying on o-anisidine system,the correlation coefficient of these two methods was-0.92.showing a good negative correlation.At the same time,it was found that the concentration of calcium carbonate had a good effect on Aspergillus niger production capacity and growth stability.It was mainly due to the effect of calcium carbonate on the morphology of the pellets.Under high calcium carbonate,the activity of GOD increased up to 50%.Finally,a high-throughput screening process of more than 2500 mutants was carried out and an evolved strain was obtained.The total GOD activity of the evolved strain was～3200 U/L,which was 146%higher than that of the original strain.The secretion ratios of GOD was 83%,32%higher than that of the original strain.Most importantly,an ultra-high-throughput screening system,based on droplet microfluidic sorting was developed and was used to improve yield of lactic acid in Bacillus coagulans.The methodology involved:(1)Generation of monodisperse double emulsion which act as picoliter-reactors for bacteria by microfluidic device.(2)A novel fluorescent sensor for pH was synthesized and a screening model for lactic acid based on fluorescent signal changing along with the broth pH in microdroplets was constructed and was used to indicate the concentration of lactic acid in the microdroplets.(3)Detection and sorting of microdroplets by flow cytometry with its unmatched sensitivity and speed.We had successfully utilized a one-step W/O/W(water in oil in water)microdroplets generator to produce monodisperse double emulsion with about 30μm in diam(～12 pL in volume),at rates～300 drops/s.The coefficient of variation of its outer and inner diameters was 2.79%and 4.2%respectively.Using this system,we screened a mutagenesis library for high lactic acid-producing Bacillus coagulans with throughputs exceeding 105 clones/h.A mutant with yield of 76 g L-1 lactic acid in flask,50%higher than its parent strain,was obtained by one round screening.In addition,the growing event from single Bacillus coagulans was tracked and quantified.In addition to establishing a practical utility of the screening system,the accessible feature of our system will also enable other screening processes for improved strains.