[Development of an LB cloning system and its application in expression of fusion genes in sp. WG].

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes Ⅰ and CⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of Ⅰ and CⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes and of sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain sp. WG/pBBR1MCS-3-LB- was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.