Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications

Abstract

The use of Bacillus subtilis in synthetic biology and metabolic engineering is highly desirable to take advantage of the unique metabolic pathways present in this organism. To do this, an evaluation of B. subtilis’ intrinsic biological parts is required to determine the best strategies to accurately regulate metabolic circuits and expression of target proteins. The strengths of promoter candidates were evaluated by measuring relative fluorescence units of a green fluorescent protein reporter, integrated into B. subtilis’ chromosome. A total of 84 predicted promoter sequences located upstream of different classes of proteins including heat shock proteins, cell-envelope proteins, and proteins resistant against toxic metals (based on similarity) and other kinds of genes were tested. The expression levels measured ranged from 0.0023 to 4.53-fold of the activity of the well-characterized strong promoter P43. No significant shifts were observed when strains, carrying different promoter candidates, were cultured at high temperature or in media with ethanol, but some strains showed increased activity when cultured under high osmotic pressure. Randomly selected promoter candidates were tested and found to activate transcription of thermostable β-galactosidase (bgaB) at a similar level, implying the ability of these sequences to function as promoter elements in multiple genetic contexts. In addition, selected promoters elevated the final production of both cytoplasmic bgaB and secreted protein α-amylase to about fourfold and twofold, respectively. The generated data allows a deeper understanding of B. subtilis’ metabolism and will facilitate future work to develop this organism for synthetic biology.

Quorum sensing inBacillus subtilisslows down biofilm formation by enabling sporulation bet hedging

“…3). P 43 is a constitutively active promoter (54), which enables the visualization of all metabolically active cells in the floating biofilm. The Δ comQ cells are less densely packed than those in the wild type floating biofilm and appear to be consisted mainly by chained cells (Figure 3), suggesting fast division.…”

“…To test this, we normalized the cumulative P epsA and P tapA transcriptional activity to the P 43 transcriptional activity. P 43 is a strong, constitutively expressed promoter (54). By doing this, we obtained a simple quantitative measure of promoter transcriptional activity in single cells with an active P 43 promoter.…”

“…The P 43 - yfp construct from Pkm3-p43- yfp plasmid (75) was digested with EcoRI and BamHI and ligated into the pSac-Cm (77) plasmid to form the pEM1071 plasmid carrying a P 43 - yfp fusion inside the sacA integration site. P 43 is a constitutively expressed promoter in Bacillus subtilis (54), located upstream from the yfp gene in the pEM1071 plasmid. This vector was then digested with HindIII and BamHI restriction enzymes to remove yfp gene from the original vector and then simultaneously ligated with the digested mKate2 fragment to construct the plasmid pMS7.…”

Fine‐tuning the (2S)‐naringenin synthetic pathway using an iterative high‐throughput balancing strategy

“…Therefore, the final pathway libraries, which carried different promoter‐gene combinations, have a small size (~50,000), for which the multiwell plate‐based screening method could already fulfill the requirement. With the development of high‐throughput sequencing technology, many native promoters from diverse industrial microorganisms, including E. coli (S. Zhou, Ding et al, ), S. cerevisiae (Redden & Alper, ; Yuan et al, ), Bacillus subtilis (Song et al, ; Yang, Du, Chen, & Kang, ), Streptomycetes (Li et al, ; Y. Luo, Zhang, Barton, & Zhao, ), and Hyperthermophilic archaeon (S. H. Lee et al, ), have been screened and characterized. These promoters provide the tools necessary to fine‐tune multigene pathways by IHTB strategy.…”

Saturation mutagenesis and self‐inducible expression of trehalose synthase in Bacillus subtilis

“…35 However, the fermentation cost was found to increase owing to the addition of the inducer. To avoid the addition of the inducer, and to simplify the operational procedure, numerous novel promoters that respond to pH, 36 oxygen, 37 temperature, [38][39][40][41][42] osmolarity, 43 and stresses to the cellular envelope 44 have also been explored. Auto-inducible expression systems have recently been constructed, [45][46][47][48] and their use has entered a new stage.…”