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

Han Xue; Hui LI; Mengqi Chen; Zaimei Zhang; Zhongrui Guo; Hu Zhu; Jiqian Wang; Yawei Sun

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

Author: Han XUE ¹ ; Hui LI ¹ ; Mengqi CHEN ¹ ; Zaimei ZHANG ¹ ; Zhongrui GUO ¹ ; Hu ZHU ² ; Jiqian WANG ¹ ; Yawei SUN ¹
Author Information

1. State Key Laboratory of Heavy Oil Processing, Department of Biological and Energy Chemical eEgineering, College of Chemistry and Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, Shandong, China.
2. Engineering Research Center of Industrial Biocatalysis, College of Chemistry and Materials Science, Fujian Normal University, Fuzhou 350007, Fujian, China.
Publication Type:Journal Article
Keywords: LB cloning system; Sphingomonas sp. WG; gene fusion; glycosyltransferase; sphingan
MeSH: Base Sequence; Cloning, Molecular; Fermentation; Plasmids/genetics*; Sphingomonas/metabolism*
From: Chinese Journal of Biotechnology 2022;38(4):1576-1588
CountryChina
Language:Chinese
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.