Engineering High Yield L-Arginine-Producing Strain Employing Enzyme Linked Fluorescent Quantitative-Single Cell Microreactor Based Ultrahigh-Throughput Screening Method

Objective: To establish an ultra-high throughput screening system based on fluorescence-activated droplet sorting for arginine-producing bacteria,and to improvethe yield of arginine-producing bacteria by directional evolution screening.Methods: The specific enzyme-linked fluorescence coupling method of arginine was established by using substrate specific arginine oxidase,and the ultra-high throughput screening system of single cell microreactor for arginine engineering bacteria was further established by using droplet microfluidic technology.The mutation library of arginine engineering bacteria was established by using ARTP mutagenesis method,and the mutant strain with improved yield was obtained by sereening with high throughput screening system.After fermentation verification,the content of L-arginine was quantitatively detected by high performance liquid chromatography.Results: 1.A stable and accurate enzyme-linked fluorescence detection system for detecting L-arginine was established,which can accurately detect 1-100 μM L-arginine.2,based on droplet microfluidic technology,the screening flux is large and the speed is fast.Through constant air pressure,the stability of droplets is guaranteed,and 10 droplets can be screened every day.3.A strain with high L-arginine yield was screened in this paper.The L-arginine yield reached 8.81 g/L,which was 1.4 times that of the original strain.Conclusion: The breeding method of arginine-producing strain based on ultrahigh-throughput screening of micro-droplet sorting activated by fluorescence has been established,which can realize the screening of engineering bacteria at single celll evel,and the screening flux is as high as 100 million reactions per day,which is more than 10,000 times higher than the traditional microtiter plate method.It has been successfully used for screening the mutation library of arginine engineering bacteria.