Construction of novel shuttle expression vectors for gene expression in <i>Bacillus subtilis</i> and <i>Bacillus pumilus</i>

Abstract

A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4. Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B. subtilis WB600. These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06. The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B. subtilis. In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome. Furthermore, pSU03-AP could also be used to transform and replicate in B. pumilus BA06 under selective pressure. In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B. pumilus.

Metabolic Engineering of Bacillus subtilis Toward Taxadiene Biosynthesis as the First Committed Step for Taxol Production

“…Segregational stability was measured by evaluating the growth of B. subtilis 168 strain harboring the pDR_txs, pBS0E_crtE, and p04_SDFHCEGA constructs in 2xYT medium without antibiotics for 30 generations involving several subcultures by adapting a previously reported protocol (Shao et al., 2015). The cells of B. subtilis 168 were first grown in 2 ml 2xYT broth containing 10 μg/ml spectinomycin, 10 μg/ml erythromycin, and 5 μg/ml chloramphenicol to select the different constructs, respectively.…”

Extracellular Proteome Profiling of Bacillus pumilus SCU11 Producing Alkaline Protease for Dehairing

“…The gram-positive endospore-forming genus Bacillus is one of the best-known industrial microorganisms for high-scale production of industrial enzymes, such as amylase, protease, and lipase. Currently, approximately 60% of industrial enzymes of the world are produced by Bacillus [43,51].…”

Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering

“…To assess the activity of 14 promoters, their upstream sequences (about 500–600 bp) from the start codon were amplified by PCR and inserted at the immediately upstream of the aprE gene encoding the alkaline protease from B. pumilus BA06 in the expression vector pSU03-AP [ 38 ]. The resulting 14 expression vectors were transformed into B. subtilis WB600 and confirmed by colony PCR.…”

“…The primers used to construct each vector were listed in Table S1 . The vector pSU03-AP allows to secretory expression of the alkaline protease (AprE) from B. pumilus BA06 in Bacillus [ 38 ]. pMUTIN4 was used to provide the lac I gene [ 39 ].…”

“…The PCR reaction contained an initial denaturation at 95 °C for 3 min, 30 cycles with denaturation at 95 °C for 15 s, annealing at 58 °C for 15 s, extension at 72 °C for 5 min. Meanwhile, the lac I gene with the promoter (P enP ) and the terminator sequence from the plasmid pMUTIN4 [ 38 ] were also amplified using two pairs of primers (PlacI.F/PlacI.R and PrrnBT-F/PrrnBT-R), respectively. These two fragments were subsequently cloned between the Amp and Kan -resistant genes in pSU03-P 2069 -GFP and pSU03-P 2069M -GFP via recombination cloning ( Figure S2B ), respectively, resulting in two expression vectors of pSU03-P 2069 -lacI-GFP and pSU03-P 2069M -lacI-GFP.…”