Enhanced Expression of Pullulanase in Bacillus subtilis by New Strong Promoters Mined From Transcriptome Data, Both Alone and in Combination

Abstract

Pullulanase plays an important role as a starch hydrolysis enzyme in the production of bio-fuels and animal feed, and in the food industry. Compared to the methods currently used for pullulanase production, synthesis by Bacillus subtilis would be safer and easier. However, the current yield of pullulanase from B. subtilis is low to meet industrial requirements. Therefore, it is necessary to improve the yield of pullulanase by B. subtilis. In this study, we mined 10 highly active promoters from B. subtilis based on transcriptome and bioinformatic data. Individual promoters and combinations of promoters were used to improve the yield of pullulanase in B. subtilis BS001. Four recombinant strains with new promoters (Phag, PtufA, PsodA, and PfusA) had higher enzyme activity than the control (PamyE). The strain containing PsodA+fusA (163 U/mL) and the strain containing PsodA+fusA+amyE (336 U/mL) had the highest activity among the analyzed dual- and triple-promoter construct stains in shake flask, which were 2.29 and 4.73 times higher than that of the strain with PamyE, respectively. Moreover, the activity of the strain containing PsodA+fusA+amyE showed a maximum activity of 1,555 U/mL, which was 21.9 times higher than that of the flask-grown PamyE strain in a 50-liter fermenter. Our work showed that these four strong promoters mined from transcriptome data and their combinations could reliably increase the yield of pullulanase in quantities suitable for industrial applications.

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

“…The strength of promoters that control the expression level of recombinant proteins is intimately related to transcriptional behavior [ 11 ]. In the past few decades, single strong promoter [ 12 , 13 ], dual-promoter [ 14 ], and triple-promoter [ 15 , 16 ] expression systems were created to improve transcriptional levels. The ribosome binding site (RBS) and spacer between the RBS and the start codon were also components that allowed gene expression to be regulated at the translational level [ 17 , 18 , 19 , 20 ].…”

The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins

“…37 These enzymes were also produced using the P sodA -fusA -amyE triple promoter. 91,92 Kang et al 46 used a system containing a P amyE-cdd dual promoter and a (Pac) signal peptide to increase the expression of a recombinant amidase (Bm-Ami). The extracellular activity of Bm-Ami containing the plasmid pBSHdd2-20 reached 10.72 U/mg À1 DCW after 52 h in a scaling fermentation that, according to the authors, was the highest secretion obtained from Bm-Ami to date.…”

“… 37 These enzymes were also produced using the P sodA - fusA - amyE triple promoter. 91 , 92 Kang et al . 46 used a system containing a P amyE-cdd dual promoter and a ( Pac ) signal peptide to increase the expression of a recombinant amidase ( Bm -Ami).…”

Advances on systems metabolic engineering of Bacillus subtilis as a chassis cell

“…Pullulanase has broad application in industry, but the amount of B. subtilis produced is low. Using transcriptome data to find the strong promoters and to increase the amount of pullulanase produced by B. subtilis [ 67 ]. Overall, systematic biology can be used to establish metabolic models, predict gene function and protein structure, and guide metabolic engineering.…”