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Abstract
BackgroundIn past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus.ResultsUsing GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP) was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA). The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds) than those obtained using the available plasmids based on the P2 constitutive promoter.ConclusionCombining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio) and B. megaterium (from Mobitec), we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.
Highlights
In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins
When using the E. coli xylose (D-glucose) isomerase (XylA) (GenBank: NP_418022) as input, no putative homologues were found in the Brevibacillus brevis NBRC 100599 genome
A BLAST search across the Brevibacillus brevis NBRC 100599 genome did not reveal any XylA and XylB homologues. These findings, coupled with the inability of Brevibacillus to grow in minimal medium with xylose as the sole carbon source, confirm that, differently from B. megaterium [25], the Brevibacillus strain used in this work is naturally deficient in xylose utilization
Summary
In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. To B. megaterium, the commercially available Brevibacillus strain does not form spores and presents limited intracellular and extracellular protease activities All these aspects together make this bacterium attractive because it is more user-friendly compared to other Gram positive microorganisms. A clear disadvantage though is that all the commercially available expression vectors designed for Brevibacillus choshinensis SP3 rely on constitutive promoters and do not allow a tightly controlled gene expression. This poses a limit to the expression of foreign proteins potentially toxic to the host cell and can impact on the final yield because of a sustained metabolic burden
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