N-acetyl-D-neuraminidase Whole-cell Catalysis And I-SceI-mediated Genome Modification Of Corynebacterium Glutamicum ATCC13032

N-acety1-D-neuraminic acid(Neu5Ac)is a natural occuing sialic acid that is often found at the terminal of sugar chain attached to the cell surface.Neu5 Ac is the chemical synthesis precursor of anti-flu medicine Zanamivir.Whole cells biotransformation of Neu5 Ac,with its feasibility and inexpensive characteritics,has been the main strategy for Neu5 Ac production.To explore the technologyof whole cells biotransformation of Neu5 Ac,we firstly cloned and expressed five N-acyl-D-glucosamine 2-epimerase(AGE)genes,and found that human gut source source Bacteroides thetaiotaomicron VPI-5482 origin BT0453 shows around 50%solubility which is the best among the AGEs.We also tried the L-arabinose and L-rhamonose inducible systems for BT0453 expression optmization,however,no obvious expression was observed for either system.Aldoase expression result showed that Escherichia coli origin nan A is almost all soluble.We then fused the BT0453 and nan A genes into one plasmid,obtabing p LS3706 A.We applied the combination of recombineering and homing endonucleqase I-Sce I-mediated double-strand break repair for the BT0453 and nan A cassette knock-in,generating LS3702.To facilate the the transformation of Neu5 Ac,we knocked in the glm S*72 gene into the nag ABE locus of E.coli BL21(DE3)genome,obtaining E.coli LS3704;knocked in the Sc GNA1 gene into the man XYZ locus of E.coli LS3704 genome,obtaining LS3706;knocked in another copy of the Sc GNA1 gene into the fuc IK locus of E.coli LS3706 genome,obtaining LS3708.Then two plasmid-based,one plasmid-based and chromosome-based Neu5 Ac biotransformation systems were assayed for the Nue5 Ac production.It turned out that Neu5 Ac molar yield of two plasmid-based and sible plasmid-based system was 50.2% and 47.7%,respectively,whereas the chromsome-based system shows the fastest transformnation reaction with the hihest molar yield reach 47.6%.Further modifications strains,nevertheless,did not show observable Neu5 Ac transformation.Corynebacterium glutamicum ATCC 13032 and its derivatoives are important amino acid producers;genome editing of the strain holds the potential for Amino acid yield improvement.Currently used methods,however,are tedious and time-consuming,while the I-Sce I-mediated method may circumvent the shortcomings.By PCR amplification of the homologous arms,cloning into T-Vector and finally cloning the suicide vector,crt I2 deletion plasmid p LS3726,CGP1 deletion plasmid p LS3728,CGP2 deletion plasmid p LS3730 CGP3 deletion plasmid p LS3732 and dkan knock-in paslmid p LS3735 were obtaibned.Homologhous recombination of one side homogous arm denerated the ingetrated strain,and under the expression of I-Sce I via IPTG induction,I-Sce I forces the second homolgos recombination,and further by PCR and sequencing screening,the target gemome modification strain could be generated.There is no medium change by the Sac B counterselection.Experiments results showed that crt operon deletion strain LS3752,CGP1 deletion strain LS3754 and dkan knock-in strain LS3760 were successfully obtained.The deletion of CGP2 and CGP3 is in progress.Based on the kanamycin repair selection of strain LS3760,comparison between leading strand oligo and lagging strans oligo showed the later is about five-fold that of former.We also found that the more the DNA amount,the higher the recombination efficiency.Phosthiolate d oligs as well as the multiple sites mutation were tested and it turned out that no multiple mutations were generated.