Secretory expression of a heterologous protein, Aiio-AIO6BS, in Bacillus subtilis via a non-classical secretion pathway

• Published in: Biochemical and biophysical research communications 2016-09, Vol.478 (2), p.881-886
• Main Authors: Pan, Xingliang, Yang, Yalin, Liu, Xuewei, Li, Dong, Li, Juan, Guo, Xiaoze, Zhou, Zhigang
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; AIO6BS; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Secretion Systems - genetics; Bacterial Secretion Systems - metabolism; Cell lysis; Codon - chemistry; Codon - metabolism; Gene Expression Regulation, Bacterial; Mutation; N-Acetylmuramoyl-L-alanine Amidase - genetics; N-Acetylmuramoyl-L-alanine Amidase - metabolism; Non-classical export pathway; Promoter Regions, Genetic; Protein Engineering; Protein Transport; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; SEC Translocation Channels - genetics; SEC Translocation Channels - metabolism; Secretion; Secretory Pathway - genetics
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2016.08.045

The quenching enzyme AIO6 (AiiO-AIO6) has been reported as a feed additive preparation for application in aquaculture and biological control of pathogenic Aeromonas hydrophila. We developed an economical strategy to express AIO6BS (AiiO-AIO6BS, codon optimized AIO6 in Bacillus subtilis) in Bacillus subtilis for facilitating its widespread application. Promoter p43 without the signal peptide was used for secretory expression of AIO6BS in B. subtilis. Western blotting analysis demonstrated that AIO6BS was successfully expressed and secreted into the cell culture. Expression analysis of AIO6BS in the single or double mutant of the lytC and lytD genes for cell autolysis in B. subtilis 1A751 and cell autolysis-resistant engineered strain LM2531 derived from the wild type 168 indicated that the release of the heterologous protein AIO6BS was not simply mediated by cell lysis. Expression level of AIO6BS did not change in the mutants of B. subtilis that harbored mutations in the secA, tatAC, or ecsA genes compared with that in the parent wild type strain. These results suggested the AIO6BS protein was likely secreted via a non-classical secretion pathway. The expression analysis of the various N- or C-terminal truncated gene products indicated that AIO6BS probably acts as an export signal to direct its self-secretion across the cell membrane. •Seven (7) N-terminus residues and 3 C-terminus residues of the AIO6BS are required for its secretion.•Glu7 mutations within AIO6BS have no impact on protein secretion.•The cys267 or hydrophobic residue of the AIO6BS is important for protein expression and secretion in Bacillus subtilis.