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Bacillus subtilis as heterologous host for the secretory production of the non-ribosomal cyclodepsipeptide enniatin.

Zobel S, Kumpfmüller J, Süssmuth RD, Schweder T - Appl. Microbiol. Biotechnol. (2014)

Bottom Line: We generated various enniatin-producing B. subtilis strains, allowing for either single chromosomal or plasmid-based multi-copy expression of the esyn cluster under the control of an acetoin-inducible promoter system.Optimization of cultivation conditions, combined with modifications of the genetic background and multi-copy plasmid-based esyn expression, resulted in a secretory production of enniatin B.This work presents B. subtilis as a suitable host for the expression of heterologous eukaryotic non-ribosomal peptide synthetases (NRPS) clusters.

View Article: PubMed Central - PubMed

Affiliation: Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623, Berlin, Germany.

ABSTRACT
The heterologous expression of genes or gene clusters in microbial hosts, followed by metabolic engineering of biosynthetic pathways, is key to access industrially and pharmaceutically relevant compounds in an economically affordable and sustainable manner. Therefore, platforms need to be developed, which provide tools for the controlled synthesis of bioactive compounds. The Gram-positive bacterium Bacillus subtilis is a promising candidate for such applications, as it is generally regarded as a safe production host, its physiology is well investigated and a variety of tools is available for its genetic manipulation. Furthermore, this industrially relevant bacterium provides a high secretory potential not only for enzymes but also for primary and secondary metabolites. In this study, we present the first heterologous expression of an eukaryotic non-ribosomal peptide synthetase gene (esyn) coding for the biosynthesis of the small molecule enniatin in B. subtilis. Enniatin is a pharmaceutically used cyclodepsipeptide for treatment of topical bacterial and fungal infections. We generated various enniatin-producing B. subtilis strains, allowing for either single chromosomal or plasmid-based multi-copy expression of the esyn cluster under the control of an acetoin-inducible promoter system. Optimization of cultivation conditions, combined with modifications of the genetic background and multi-copy plasmid-based esyn expression, resulted in a secretory production of enniatin B. This work presents B. subtilis as a suitable host for the expression of heterologous eukaryotic non-ribosomal peptide synthetases (NRPS) clusters.

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Extra-chromosomal multi-copy expression of esyn in B. subtilis. Expression of the esyn gene on the multi-copy plasmid pJK255 (with ∼200 copies) in BsJK106 in comparison to BsSZ10 leads to 130-fold increased enniatin level in the supernatant. N = 4
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Fig5: Extra-chromosomal multi-copy expression of esyn in B. subtilis. Expression of the esyn gene on the multi-copy plasmid pJK255 (with ∼200 copies) in BsJK106 in comparison to BsSZ10 leads to 130-fold increased enniatin level in the supernatant. N = 4

Mentions: We furthermore tested the effect of the cloning of the esyn cluster into the high-copy plasmid pMSE3. The resulting strain BsJK106 (pJK255), which possesses approximately 200 copies of the esyn gene (Fig. S8), produced the highest yield of enniatin (Fig. 5). We quantified the extracts of supernatant of BsJK106 cultures in comparison to an external enniatin standard and measured yields of 1.1 mg/L enniatin under optimized cultivation conditions. An additional gene copy of the positive transcription factors sigL and acoR (Ali et al. 2001; Kabisch et al. 2013b) did not lead to a further enhanced enniatin production (data not shown).Fig. 5


Bacillus subtilis as heterologous host for the secretory production of the non-ribosomal cyclodepsipeptide enniatin.

Zobel S, Kumpfmüller J, Süssmuth RD, Schweder T - Appl. Microbiol. Biotechnol. (2014)

Extra-chromosomal multi-copy expression of esyn in B. subtilis. Expression of the esyn gene on the multi-copy plasmid pJK255 (with ∼200 copies) in BsJK106 in comparison to BsSZ10 leads to 130-fold increased enniatin level in the supernatant. N = 4
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4306738&req=5

Fig5: Extra-chromosomal multi-copy expression of esyn in B. subtilis. Expression of the esyn gene on the multi-copy plasmid pJK255 (with ∼200 copies) in BsJK106 in comparison to BsSZ10 leads to 130-fold increased enniatin level in the supernatant. N = 4
Mentions: We furthermore tested the effect of the cloning of the esyn cluster into the high-copy plasmid pMSE3. The resulting strain BsJK106 (pJK255), which possesses approximately 200 copies of the esyn gene (Fig. S8), produced the highest yield of enniatin (Fig. 5). We quantified the extracts of supernatant of BsJK106 cultures in comparison to an external enniatin standard and measured yields of 1.1 mg/L enniatin under optimized cultivation conditions. An additional gene copy of the positive transcription factors sigL and acoR (Ali et al. 2001; Kabisch et al. 2013b) did not lead to a further enhanced enniatin production (data not shown).Fig. 5

Bottom Line: We generated various enniatin-producing B. subtilis strains, allowing for either single chromosomal or plasmid-based multi-copy expression of the esyn cluster under the control of an acetoin-inducible promoter system.Optimization of cultivation conditions, combined with modifications of the genetic background and multi-copy plasmid-based esyn expression, resulted in a secretory production of enniatin B.This work presents B. subtilis as a suitable host for the expression of heterologous eukaryotic non-ribosomal peptide synthetases (NRPS) clusters.

View Article: PubMed Central - PubMed

Affiliation: Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623, Berlin, Germany.

ABSTRACT
The heterologous expression of genes or gene clusters in microbial hosts, followed by metabolic engineering of biosynthetic pathways, is key to access industrially and pharmaceutically relevant compounds in an economically affordable and sustainable manner. Therefore, platforms need to be developed, which provide tools for the controlled synthesis of bioactive compounds. The Gram-positive bacterium Bacillus subtilis is a promising candidate for such applications, as it is generally regarded as a safe production host, its physiology is well investigated and a variety of tools is available for its genetic manipulation. Furthermore, this industrially relevant bacterium provides a high secretory potential not only for enzymes but also for primary and secondary metabolites. In this study, we present the first heterologous expression of an eukaryotic non-ribosomal peptide synthetase gene (esyn) coding for the biosynthesis of the small molecule enniatin in B. subtilis. Enniatin is a pharmaceutically used cyclodepsipeptide for treatment of topical bacterial and fungal infections. We generated various enniatin-producing B. subtilis strains, allowing for either single chromosomal or plasmid-based multi-copy expression of the esyn cluster under the control of an acetoin-inducible promoter system. Optimization of cultivation conditions, combined with modifications of the genetic background and multi-copy plasmid-based esyn expression, resulted in a secretory production of enniatin B. This work presents B. subtilis as a suitable host for the expression of heterologous eukaryotic non-ribosomal peptide synthetases (NRPS) clusters.

Show MeSH
Related in: MedlinePlus