Region Dependent Efficiency for Recombinational Transfer of the<i>Bacillus subtilis</i>168 Genome

Tomita, Satoshi; Tsuge, Kenji; Kikuchi, Yo; Itaya, Mitsuhiro

doi:10.1271/bbb.68.1382

Cited by 8 publications

(4 citation statements)

References 7 publications

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“…Deletion of this cluster furthermore reduces the genome by 76.5 kb, which means less metabolic cost for replication and unnecessary protein production. It has already been shown elsewhere, that efficiency of recombinational transfer may be region dependent (Tomita et al, 2004a) . The integration of completely synthetic and unique regions into the chromosome could be a way to address this issue.…”

Section: Discussionmentioning

confidence: 83%

CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

Nadler

Bracharz

Kabisch

2018

Preprint

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The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis , we have developed an automatable system for the tuning of heterologous gene expression based on the host's intrinsic natural competence and homologous recombination capabilities. By supplying our reporter strains with a linearized, low copy number plasmid containing homology regions left and right of the reporter genes and an antibiotic resistance marker, we could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful recombination and thus circularization of our plasmid. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher.

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Section: Discussionmentioning

confidence: 83%

CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

Nadler

Bracharz

Kabisch

2018

Preprint

View full text Add to dashboard Cite

show abstract

“…Deletion of this cluster furthermore reduces the genome by 76.5 kb, which means less metabolic cost for replication and unnecessary protein production. It has already been shown elsewhere, that efficiency of recombinational transfer may be region-dependent (Tomita et al, 2004a). The integration of completely synthetic and unique regions into the chromosome could be a way to address this issue.…”

Section: Discussionmentioning

confidence: 84%

CopySwitch—in vivo Optimization of Gene Copy Numbers for Heterologous Gene Expression in Bacillus subtilis

Nadler

Bracharz

Kabisch

2019

Front. Bioeng. Biotechnol.

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The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. While there are numerous ways of doing so on protein or mRNA level, our approach aims for the underlying number of coding sequences. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the host's intrinsic natural competence and homologous recombination capabilities. Strains are transformed with a linearized, low copy number plasmid containing an antibiotic resistance marker and homology regions up- and downstream of the gene of interest. Said gene is copied onto the vector, rendering it circular and replicative and thus selectable. We could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful transformation. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher. A major benefit of this method is the ability to work with very large regions of interest, since all relevant steps are carried out in vivo and are thus far less prone to mechanical DNA damage.

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“…These are the first recombinant genomes shown to be convertible to another host [23]. The BReT system has been widely used to retrieve certain B. subtilis genome regions due to technical simplicity [71,72].…”

Section: Retrieval By Copying the Segment Of The B Subtilis Genomementioning

confidence: 99%

Recombinant Genomes: Novel Resources for Systems Biology and Synthetic Biology

Itaya

2009

Systems Biology and Synthetic Biology

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Region Dependent Efficiency for Recombinational Transfer of theBacillus subtilis168 Genome

Cited by 8 publications

References 7 publications

CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

CopySwitch -in vivooptimization of gene copy numbers for heterologous gene expression inBacillus subtilis

CopySwitch—in vivo Optimization of Gene Copy Numbers for Heterologous Gene Expression in Bacillus subtilis

Recombinant Genomes: Novel Resources for Systems Biology and Synthetic Biology

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