The Construction And Application Of A High-throughput Screening Method For Improving Lysine-producing Strains

Corynebacterium glutamicum,a non-pathogenic Corynebacterium,has been applied worldwidely for producing various amino acids,such as L-lysine.L-lysine is one of the eight essential amino acids for humans and animals,and has been extensively used in healthcare,foods and in particular animal feeds.The production performance of the lysine-producing strains is critical for the market competitiveness of the lysine industry.In this study,we constructed a new high-throughput screening method using a lysine-inducible promoter and associated signaling genes in the industrial lysine-producer C.glutamicum strains.(1)Construction of the lysine biosensors.Lysine biosensors were constructed by using a lysine inducible promoter P4 or a regulator lysG that can respond to lysine signal.Signaling genes including a yellow fluorescent gene(yfp)or a tetracycline resistant gene(tet)were regulated by P4 or lysG,respectively.(2)Evaluation and application of the lysine biosensors.Strains with different levels of lysine-producing abilities were constructed to test the response linearity of the sensors.The strains could be distinguished by YFP signal or tetracycline resistance.A high-throughput screening method was constructed accordingly using tetracycline resistant ability as a marker of lysine-producing level.Using this method,a lot of strains producing higher concentration of lysine were obtained from a mutant library of the starting strain ZL from large scale industrial production.The best five strains were named as ZL-1,ZL-2,ZL-3,ZL-4 and ZL-5.(3)After fed-batch fermentation for 60 h,the lysine yield and productivity of initial strain ZL were 29.4%(glucose/lysine,W/W)and 7 g/L,respectively,while those of ZL-1 were 40%and 8.3 g/L,respectively,ZL-2 were 38.2%and 8.1 g/L,respectively,ZL-3 were 44.6%and 7.9 g/L,respectively,ZL-4 were 39%and 8.5 g/L,respectively,and ZL-5 were 36%and 8.5 g/L respectively.The yield and productivity are improved by 22.4-51.7%and 12.8-21.4%,respectively.All of these five strains have much higher yields and productivities than the original strain ZL.The new high-throughput screening method based on lysine biosensor for the high lysine-producing C.glutamicum strain is highly efficient and useful in continuous improvement of the strains used in lysine industry.This methods may also be helpful for the strain development of other amino acid industry.