Dataset Information

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis.

ABSTRACT:

Background

The bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq.

Results

Using the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%.

Conclusions

Overproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms.

SUBMITTER: Wang B

PROVIDER: S-EPMC10633939 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Publications

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis.

Wang Biwen B van der Kloet Frans F Hamoen Leendert W LW

Microbial cell factories 20231109 1

<h4>Background</h4>The bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome ...[more]

PMID: 37946188

Similar Datasets

Project description:BackgroundBacillus subtilis is widely used for industrial enzyme production due to its capacity to efficiently secrete proteins. However, secretion efficiency of enzymes varies widely, and optimizing secretion is crucial to make production commercially viable. Previously, we have shown that overexpression of the xylanase XynA lowers expression of Clp protein chaperones, and that inactivation of CtsR, which regulates and represses clp transcription, increases the production of XynA. In the current study, we examined whether the same is the case for overexpression of the α-amylase AmyM from Geobacillus stearothermophilus by B. subtilis, and why XynA shows a different timing of secretion compared to AmyM.ResultsTranscriptome analyses revealed that B. subtilis cells overexpressing AmyM exhibited a distinct profile compared to XynA overexpressing cells, however there were also similarities and in both cases expression of CtsR controlled genes was downregulated. In contrast to XynA, inactivation of CtsR did not improve AmyM production. Upregulation of other protein chaperones, including GroEL/ES and DnaJ/K, by inactivating their transcriptional repressor HrcA, had almost no effect on XynA yields and in fact considerably lowered that of AmyM. Despite using the same promoter, the production of XynA peaks well before AmyM reaches its optimal secretion rate. Transcriptome and ribosome profiling indicated that this is neither related to transcription nor to translation regulation. We show that the reduced secretion in the stationary phase is partially due to the activity of secreted proteases, but also due to the activity of the intracellular protease LonA. The absence of this protein resulted in a 140% and 20% increased production for XynA and AmyM, respectively.ConclusionThe combination of transcriptome and ribosome profiling offered important information to determine at which cellular level production bottlenecks occurred. This helped us to identify LonA protease as an important factor influencing enzyme production yields in B. subtilis.

Dataset Information

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis.

Background

Results

Conclusions

Publications

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis.

Similar Datasets

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