Construction Of The ARG-ON Biosensor And Application In The Screening Of L-arginine Hyperproducing Strain

In recent years,biosensors have become an effective tool to obtain high yield strains from the high throughput screening of mutant strains.In this study,based on the strain of Corynebacterium crenatum SYPA5-5,the biosensor of ARG-ON?ARG-sacB and ARG-gfp?was constructed.To obtain the L-arginine high yield strains,the strain of C.crenatum SYPA5-5 which contained the biosensor was mutagenized,and the L-arginine hyperproducing strain was screened by the biosensor.The main research results are shown as follows:?1?Construction of the biosensor of ARG-ON?ARG-sacB and ARG-gfp?with the reporter gene of sacB and gfp.To construct the plasmids of PC-sacB-10 and PC-gfp-10,the PargC promoter was linked with sacB and gfp by overlap extension PCR,then the PCR products were ligated into the pDXW-10 plasmid.Finally,the argR gene was ligated into the PC-sacB-10 and PC-gfp-10plasmids,and the biosensor of ARG-ON?ARG-sacB and ARG-gfp?was constructed successfully.?2?Verification of the relationship between the fluorescence intensity or cell growth and the L-arginine production in ARG-ON?ARG-sacB and ARG-gfp?biosensor.Different L-arginine concentrations(0,20,40,60,80,100 mmol·L^-1)were added to the medium,then the strains which contained biosensors of ARG-sacB and ARG-gfp were cultivated.As can be seen from the results,the strain will grow better with the increase of L-arginine concentration,and the fluorescence intensity will become weaker with the increase of L-arginine concentration.Through the quantitative real-time PCR?qRT-PCR?analysis,it could be found that the transcription levels of reporter genes were decreased with the increase of L-arginine concentration.Subsequently,strains of C.crenatum SYPA5-5 which contained the biosensor of ARG-sacB and ARG-gfp were mutagenized,respectively,then the mutant cells were sorted by the sucrose screening plate and flow cytometry.The strains with different colony size and fluorescence intensity were screened,and the L-arginine yields of the strains were determined.As can be seen from the results,for the biosensor of ARG-sacB,the L-arginine production of the mutant strains with a small colony were low,while the L-arginine production of the mutant strains with a large colony were high.For the biosensor of ARG-gfp,the L-arginine production of the mutant strains with low fluorescence were high,while the L-arginine production of the mutant strains with high fluorescence were low.?3?High-throughput screening of the L-arginine hyperproducing strains by the biosensor of ARG-sacB with the reporter gene of sacB.Strains of C.crenatum SYPA5-5 and its system metabolic engineered strain Cc4 were mutagenized respectively,both of the strains contained the biosensor of ARG-sacB.The mutant cells were spreaded on sucrose screening plates,and the mutant strains with a large colony were selected and cultivated in 24-deep-well plates.For the strain of C.crenatum SYPA5-5,there were a total of 375 large strains on sucrose screening plates.Then,the 375 large strains were cultivated in 24-deep-well plates.Through the screening of 24-deep-well plate,14 mutant strains were obtained which exhibited a 20%increase in L-arginine production.Through the screening of shake-flask fermentation,L-arginine hyperproducing strain of GArg5 was obtained.Finally,the L-arginine yield of strain GArg5 in a 5 L bioreactor reached 64.5 g·L^-1,which was increased by 43.0%.For the strain of system metabolic engineered strain Cc4,there were a total of 189 large strains on sucrose screening plates.Then,the 189 large strains were cultivated in24-deep-well plates.Through the screening of 24-deep-well plate,11 mutant strains were obtained which exhibited a 20%increase in L-arginine production.Through the screening of shake-flask fermentation,L-arginine hyperproducing strain of SArg55 was obtained.Finally,the L-arginine yield of strain SArg55 in a 5 L bioreactor reached 81.3 g·L-1,which was increased by 18.3%.