A dual protein expression system in Bacillus subtilis

Abstract

We have developed a dual expression system for the simultaneous overexpression of two proteins in Bacillus subtilis. Two candidate genes, xylanase (xynA) and glucanase (bglS) from B. subtilis strain 168, which were engineered with independent Shine-Dalgarno (SD) sequences, were cloned in tandem into a transfer vector, which was then transformed into B. subtilis strain 1A304 (phi105MU331). The genes were under the transcriptional control of a strong promoter of a bacteriophage, phi105, where transcription was initiated upon thermal induction. Six constructs were made to compare the factors that affected the yields of the gene products. The expression level of each candidate gene was found to correspond to its position relative to the phage promoter, irrespective of the identity of the insert. The lower expression level of the second insert might have been due to limited resources for protein synthesis, a short half-life of the mRNA, or an early termination of the RNA polymerase. Curiously, gene duplications in tandem did not lead to further increase in production.

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

“…31 Although the integrative plasmid had advantages after integration into the chromosome, the expression levels were usually low. 32 The mutants obtained in this study were not affected by plasmid stability, did not need added antibiotics in the culture, and produced high amounts of c-PGA in high density medium.…”

Interaction of a Putative Transcriptional Regulatory Protein and the Thermo-inducible cts-52 Mutant Repressor in the Bacillus subtilis Phage φ105 Genome

“…The genetic switch results from a mutation (cts-52) on the wtcf105 that renders the expression system thermo-inducible. 16 -18 As shown in previous studies, 19,20 induction of heterologous gene expression in the f105MU331 system was achieved by a temperature shift from 37 8C to 50 8C. The aim of our study was to examine whether this transcriptional control is closely regulated by altered affinities between the mutant repressor and the six operators at different temperatures.…”

“…by the promoter of the holin gene, which is located 10.6 kb from the imm F region. Although thermoinduction was found to turn on the promoter activity of the holin gene, 19 it was deemed unlikely that mtcf105 or ORF4 had any direct interaction with this promoter, as no consensus operator sequence was identified at its upstream region. It is supposed that the binding of mtcf105 to certain operator sequences in the f105 genome will block either the expression of ORF4 or other trans factor necessary for triggering heterologous gene expression.…”

Complete Genome Sequence of Bacillus subtilis Phage φ105

“…Bacteriophage φ105, a well-studied temperate phage of Bacillus subtilis (1, 2), is capable of specialized transduction and has been developed into a system for cloning and expressing genes in this host (3, 4). Here I describe the complete genome resequencing of φ105.…”