| Microorganisms in nature are extremely rich and diverse.As a treasure house of human resources,microorganisms and their metabolites play a very important role in various application fields such as medicine and environmental protection.The selection and breeding of excellent strains are the soul of industrial microbial fermentation production,and the selection of strains faces two important problems:one is the difficulty of transforming good strains;the second is the cumbersome and time-consuming process of strain screening.In addition to genetic engineering breeding based on the understanding of the genetic background of the strains,traditional mutagenesis methods are more suitable for strains with unclear genetic backgrounds,complex metabolic regulation pathways,complex morphological differentiation of bacterial cells,or incomplete genetic manipulation systems.Through scientific methods,the survival rate and mutation rate of strains in these simple and direct traditional mutagenesis methods can be improved,and to a certain extent,the two problems of high strain lethality and low mutation rate in breeding work can be solved,which will help to obtain bacterial strains.provides more possibilities.In addition,among a wide variety of microbial metabolites,most microbial metabolites do not have significant properties such as ultraviolet absorption,luminescence,and fluorescence,and they often need to be identified by large-scale separation and analysis instruments,checkout presents difficulties.In recent years,a new method for the identification and detection of target products in a single cell by using microbial live cell metabolite biosensors has shown a good application prospect in the field of high-throughput screening of industrial microorganisms.Based on the previous work of our laboratory,this paper further optimized and verified the optimization of the cell-penetrating peptide（CPP）mediated FAM-aptamer Molecular becon（FAM-TC-BHQ1）Streptomyces aureus（S.aureofaciens）live cell delivery and target detection system,which improved the delivery efficiency from 3‰to 10%;at the same time,the positive correlation between the fluorescence detection signal of the system and the intracellular target content was verified,which laid a foundation for the development of microbial metabolite biosensors based on CPP carried nucleic acid probes for target-specific detection.At the same time,in order to improve the survival rate and mutation rate of strains in mutagenesis breeding,we have developed an assisted mutagenesis method that is suitable for different types of microbial cells and mediated by T4 DNA ligase into cells via penetrating peptides.The experimental results showed that the T4 DNA ligase mediated by（KH）9-BP100 was introduced into S.aureofaciens and Escherichia coli（E.coli）M-4 cells,which could improve the survival rate of bacteria in UV mutagenesis.This method can increase the mutation rate of E.coli M-4 rps L gene after UV mutagenesis by more than one order of magnitude.Therefore,the mutagenesis-assisted method constructed in this paper is expected to contribute to improving the survival rate and mutation rate of microbial strains in the mutagenesis of microbial strains.Based on the mutagenesis-assisted method constructed in this paper,we carried out UV mutagenesis of E.coli M-4 strain and screened strains with high tryptophan production.After screening,the strain E.coli MCU with the highest tryptophan yield was obtained.The tryptophan yield increased from 1.21 g/L at the beginning to 2.13 g/L,and the tryptophan content increased by 77.5%.Through the subsequent optimization of fermentation conditions and fermentation medium,the yield of tryptophan in shake flask fermentation can reach 2.56 g/L,which is 113.33%higher than the initial tryptophan content.The genetic stability test showed that the tryptophan content of the strain was 2.51g/L after 7 generations,and the genetic stability was good. |
