LB克隆系统的构建及在鞘氨醇单胞菌基因融合表达中的应用

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

摘要:

为摆脱限制性酶切位点不足的限制,构建可灵活改变多基因融合方向的表达载体,基于IIS型和IIT型限制性内切酶LguⅠ和BbvCⅠ设计开发了LB克隆系统。该克隆系统是以广宿主质粒pBBR1MCS-3为初始载体,利用PCR的方法,在其多克隆位点区插入LB片段(GCTCTTCCTCAGC)构建得到的。LB片段含LguⅠ和BbvCⅠ部分重叠识别位点,经这两种限制性内切酶酶切后可以产生相同非回文序列,利用这一性质可快速、灵活地将多个基因逐步、定向插入表达载体。为验证该克隆系统的有效性,将鞘氨醇单胞菌(Sphingomonas sp.) WG中两个糖基转移酶基因welB、welK逐步定向融合至LB克隆载体,并将重组质粒转入鞘氨醇单胞菌中表达。结果表明,基因融合表达对鞘氨醇胶产量影响较小,但是,对鞘氨醇胶粘度有重要影响。在发酵84 h时,重组菌株Sphingomonas sp.WG/pBBR1MCS-3-LB-welKB的发酵液粘度较野生株提高约24.7%,粘度提升将有助于该鞘氨醇胶在石油开采、食品等多个领域的应用。综上所述,以鞘氨醇单胞菌为研究对象,验证了LB克隆系统在多基因融合中的应用,为构建融合酶提供了一种高效的方法。

Abstract:

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 LguⅠ and BbvCⅠ.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 LguⅠ and BbvCⅠ.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 welB and welK of Sphingomonas sp.WG were consecutively fused to the LB cloning vector,and the recombinant plasmid was transferred into Sphingomonas 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 Sphingomonas sp.WG/pBBR1MCS-3-LB-welKB 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 Sphingomonas sp.WG.The LB cloning system may provide an efficient tool for fusion expression of target genes.