Establishment Of Negative Screening Marker For New Reorganization Project And Optimization Of N - Acetyl - D - Neuraminidase - Catalyzed Strain

Recombineering is a homologous recombination mediated genetic engineering. It relies on in vivo expression of homologous recombination enzymes; and is an efficient method for in vivo genetic engineering on the chromosome. Recombineering makes it possible to modify the target DNA molecule at any sites without restrictions of restriction enzyme sites. It shows the advantages of simplicity, time-saving and high recombination efficiency, and it is widely used in the gene knockout, point mutation, etc.The counterselection marker, contrary to the antibiotic resistance genes as positive selection markers, means microorganism cannot survive with its expression, and is widely used in gene cloning and DNA modification. This paper presents the results that inducible ccdB gene as a counterselection marker. Compared with the counterselection markers such as galK, thyA, sacB, tolC, it is reported that ccdB counterselection is most efficient. ccdB gene originates of F plasmid and shows the toxic effect by binding the DNA topoisomerase Ⅱ.In this study, we combined the use of antibiotic resistance gene and inducible ccdB gene under the control of plac promoter which is IPTG inducible. Inducible ccdB gene as a counterselection marker, only need to add inducer can be screened, but also can be achieved without tolerance of CcdB toxic protein modification of the strains. To knock-in foreign gene in the lacZ locus of E. coli DH10B,50 bp homologous arm flanked HA1-aacCl-plac-ccdB-HA2 gene cassette was generated through PCR, followed by introducing into the homologous region of E. coli DH10B, obtaining mutant strain containing inducible ccdB gene. After confirmation, the inducible ccdB was used as the screening marker, mKan, bla* and oligonucleotide were all successfully knocked into the target region, obtaining corresponding mutants. Under the screening of the inducible ccdB gene, pir116 gene was also successfully knocked into the lacZ region of E. coli DH10B. pirl 16 renders the R6K replicon-based plasmid high copy number, which may substitute the BUN20 host strain.The recombinase gene are reside in mutant strain, which can be induced for gene expression by adding L-arabinose, and homologous recombination happen between the linear DNA fragments, or between linear DNA and cyclic plasmids. Following the same strategy, the I-Scel gene was knocked into the uidA region of MG1655, reaching a recombination efficiency of 67.5%. I-SceI enzyme is used for double-strand break repair. Compared with plasmid based I-Scel gene, the generated strain is more stable, and is of great significance for knocking out resistance screening marker.Neu5Ac and its derivatives are important biological active molecules. These sugar molecules are often reside at the end of macromolecules and have important biological functions and medical values. For synthesizing Neu5Ac, whole-cell catalysis, because of its simple operation, low cost, environmental friendly, is superior to other methods. In this study, chromosome-based Neu5Ac-catalysing strain LS2802 was optimized by the recombinant engineering to reduce intracellular GlcNAc enzymes involved in nagABE, manXYZ and fucIK knockout, meanwhile Neu5Ac related genes glmS*72 and ScGNAl were integrated into the genome, we obtained a series of production neuraminic acid strains.