Complex Mutagenesis,Process Optimization And Mutation Gene Analysis Of Polysialic Acid Producing Strain

Polysialic acid(PSA)is a homomer of n-acetylneuraminic acid(Neu5Ac)connected directly or indirectly by alpha-2,8 and/or alpha-2,9 glucoside bonds.It exists in the body of mammals,and attaches in the form of capsule on the surface of bacteria.PSA has no immunogenicity and good biodegradability,making it an ideal carrier for drug sustained release materials.Due to the low level of PSA fermentation at present,the cost of industrial large-scale production of PSA is high,which limits the application of PSA.In this study,Escherichia coli K2 was used as the original strain.First,combined with the latest mutagenesis technology and metabolic engineering technology,engineering bacteria with high PSA yield were cultivated,and the high yield mechanism was studied at the genome and transcriptome level.Specific work is as follows:(1)By combining UV and ARTP mutagenesis of the original E.coli K2,fermentation experiments and genetic stability tests,a high yield and stable genetic PSA mutant strain was finally screened.The yield reached 0.731 g/L,which was 2.1times that of the original E.coli.We’ll name it E.coli A29.(2)The fermentation medium and culture conditions of the selected mutant E.coli A29 were optimized at the shaking flask level,including carbon source,nitrogen source,phosphate,p H,inoculation amount,fermentation time,culture temperature,etc.After the optimal medium conditions were obtained,the mutagenesis strain E.coli A29 was subjected to high efficiency fermentation in a 7 L fermenter.The PSA yield reached 6.09 g/L,which was 44.67% higher than that of the unoptimized strain.The fermentation efficiency of the mutagenesis strain was successfully improved.(3)Combined with comparative genomics and transcriptomics,the original E.coli K2 and mutant E.coli A29 were analyzed.The comparative analysis showed that there were significant differences in E.coli A29 genes,including three genes that were significantly up-regulated and two genes that were significantly down-regulated.These differentially expressed genes were mainly involved in the transport and synthesis of related enzymes,AAA-ATPase transport and catalysis,and cell biofilm formation and repair.With the help of omics analysis technology,the influence of different gene expression levels on the metabolic pathway of PSA biosynthesis was explained,and several important clues related to PSA high yield were excavated,laying a theoretical foundation for future work.(4)CRISPR-CAS9 system was used to knock out the Neu5 Ac encoding gene nan ATEK of E.coli K2 and construct E.coli K2Δnan ATEK.Colony PCR verification and DNA sequencing were used to determine the success of nan ATKE gene knockout.E.coli K2Δnan ATEK showed that the PSA synthesis ability increased significantly from 0.361 g/L to 0.634 g/L,compared with E.coli K2.The knockout of nan ATEK gene can promote PSA synthesis,which provides a basis for further study of PSA high yield theory.