Improved Expression and Optimization of Trehalose Synthase by Regulation of Pglv in Bacillus subtilis

Hongling Liu,Shaojie Yang,Ruiming Wang,Hao Liu,Tengfei Wang

doi:10.1038/s41598-019-43172-z

Abstract

Trehalose synthase (TreS) converts maltose to trehalose, which has several important functions; therefore, enhancing TreS expression is desirable. Here, a recombinant Bacillus subtilis W800N (ΔamyE)-Pglv strain was constructed to achieve enhanced expression of TreS. Process optimization strategies were developed to improve the expression level of TreS in B. subtilis W800N (ΔamyE)-Pglv. Intracellular activity of TreS was induced using 60 g/L of maltose in shake flask culture. The protein activity reached 5211 ± 134 U/g at 33 °C and pH 7.0 in Luria-Bertani medium. A fed-batch fermentation strategy was applied in a 30 L fermenter containing 18 L terrific broth to achieve high cell density by replacing glycerol with high maltose syrup as a carbon source and an inducer. After 32 h of fermentation, recombinant B. subtilis W800N (ΔamyE)-Pglv activity reached 6850 ± 287 U/g dry cell weight. Our results demonstrate the efficiency of the Pglv promoter in increasing the expression of TreS in B. subtilis W800N (ΔamyE)-Pglv.

Highlights

Trehalose is a non-reducing disaccharide comprising two glucose units joined by an α-1,1-glycosidic bond[1]
The recombinant clones (B. subtilis W800N (ΔamyE)-promoter of glv operon (Pglv), M1; B. subtilis W800N-Pglv, M2; and B. subtilis W800N (ΔamyE)-Pgrac, M3) were subjected to fermentation followed by sonication to extract intracellular proteins
The recombinant proteins had a molecular weight of nearly 75 kDa (Fig. 2), consistent with the molecular weight deduced from the amino acid sequence of TreS. These data suggest that the Pglv promoter was superior to Pgrac promoter in enhancing TreS expression and that the knockout of amyE gene further increased the expression of TreS in the recombinant group

Summary

Introduction

Trehalose is a non-reducing disaccharide comprising two glucose units joined by an α-1,1-glycosidic bond[1]. The drawbacks of the dual-enzymatic method are that it requires the cultivation of two types of recombinant bacteria. TreS is produced by several strains of bacteria, including Bacillus licheniformis, Thermobifida fusca, and Yarrowia lipolytica, which have been considered for use in trehalose production. Genetic engineering of bacteria has been carried out with the goal of improving the expression of TreS, including cloning of treS gene in E. coli[11,12,13,14]. This approach is time-consuming, and the removal of pathogenic enterobacteria prior to the use of trehalose in the food industry is an expensive process. Strains E. coli DH5α B. subtilis 168 B. subtilis W800N Plasmids pHT01 pHT01-Pglv-treS pHT01-Pglv-treS Primers Pglv-1-F Pglv-1-R-C Pglv-2-F-C Pglv-2-R-C TreS-F-C TreS-R Spe-F Spe-R amyE-1-F amyE-1-R

Methods

Results

Discussion

Conclusion