Induction of CtsR-regulated chaperones improves Xylanase production in Bacillus subtilis

We examined the effect of high-level expression of the commercially important endo-1,4-ß-xylanase XynA on the B. subtilis transcriptome using RNA-seq. Rather unexpectedly, we found a reduced expression of several protein chaperones, including ClpC, ClpE and ClpX, was downregulated when XynA was overproduced. Expression of these proteins is controlled by the transcriptional repressor CtsR. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. Preventing this downregulation by knocking out the involved transcriptional repressor CtsR resulted in increased XynA production by more than 25 %. Overall design: We used B. subtilis 168 derivative BSB1, which is prototrophic for tryptophan, and removed the native xynA gene through a marker-free clean deletion procedure. This was to assure that the negative control strain does not produce any XynA. Overexpression of XynA was achieved by cloning xynA in the multicopy expression plasmid pUB110 behind the strong amyQ promoter. As a negative control, we used the same plasmid lacking the xynA gene. The strains were grown in nutrient-rich LB medium at 37 °C in the presence of 50 µg/ml kanamycin to maintain the plasmids. We sampled cells at 3 h (OD~0.8) and 6h growth (OD~4) and performed RNA-seq. We compared the transcriptomes between XynA overproducing cells and non-producing cells. Two independent biological replicates were performed.

Identifier
Source https://data.blue-cloud.org/search-details?step=~012FD10FAD911A18F4DD0A72385676D1F38F8F7BC4E
Metadata Access https://data.blue-cloud.org/api/collections/FD10FAD911A18F4DD0A72385676D1F38F8F7BC4E
Provenance
Instrument NextSeq 550; ILLUMINA
Publisher Blue-Cloud Data Discovery & Access service; ELIXIR-ENA
Contributor Bacterial Cell Biology, Swammerdam Institute for Life Science, University of Amsterdam
Publication Year 2024
OpenAccess true
Contact blue-cloud-support(at)maris.nl
Representation
Discipline Marine Science
Temporal Point 2023-02-27T00:00:00Z