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

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.

• Publication year

  2014

• Venue

  Applied Microbiology and Biotechnology

• Publication date

  2014-11-15

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1007/s00253-014-6199-0PMID 25398283PMCID 4306738
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization, yield and activity

  2014cited by this paper
• Reconstituted biosynthesis of the nonribosomal macrolactone antibiotic valinomycin in Escherichia coli.

  2014cited by this paper
• Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production

  2014cited by this paper
• Characterization and optimization of Bacillus subtilis ATCC 6051 as an expression host.

  2013influential reference
• An optimized technique for rapid genome modifications of Bacillus subtilis.

  2013influential reference
• Metabolic engineering of Bacillus subtilis for growth on overflow metabolites

  2013influential reference
• Classification of Bacillus beneficial substances related to plants, humans and animals.

  2012cited by this paper
• In vitro chemoenzymatic and in vivo biocatalytic syntheses of new beauvericin analogues.

  2012influential reference
• Fed‐batch culture of Escherichia coli for L‐valine production based on in silico flux response analysis

  2011cited by this paper
• Tetracycline Modulates the Valine Induced Stringent Response and Decreases Expressed RpoS in Escherichia coli

  2011cited by this paper
• Fungal cyclooligomer depsipeptides: from classical biochemistry to combinatorial biosynthesis.

  2011influential reference
• ggplot2: Elegant Graphics for Data Analysis

  2010cited by this paper
• Qualified presumption of safety (QPS): a generic risk assessment approach for biological agents notified to the European Food Safety Authority (EFSA)

  2010cited by this paper
• In vitro Synthesis of New Cyclodepsipeptides of the PF1022‐Type: Probing the α‐D‐Hydroxy Acid Tolerance of PF1022 Synthetase

  2009cited by this paper
• Identification of a Polymyxin Synthetase Gene Cluster of Paenibacillus polymyxa and Heterologous Expression of the Gene in Bacillus subtilis

  2009cited by this paper
• Cre/lox System and PCR-Based Genome Engineering in Bacillus subtilis

  2008cited by this paper
• The Origins of 168, W23, and Other Bacillus subtilis Legacy Strains

  2008cited by this paper
• Targeting the Bacillus subtilis genome: an efficient and clean method for gene disruption.

  2007cited by this paper
• Comparative analysis of the complete genome sequence of the plant growth–promoting bacterium Bacillus amyloliquefaciens FZB42

  2007cited by this paper
• The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis

  2007cited by this paper
• Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC

  2007cited by this paper
• Production of the non-ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment.

  2007cited by this paper
• In Vitro Synthesis of New Enniatins: Probing the α‐D‐Hydroxy Carboxylic Acid Binding Pocket of the Multienzyme Enniatin Synthetase

  2007cited by this paper
• Enniatin exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells.

  2007cited by this paper
• An acetoin-regulated expression system of Bacillussubtilis

  2006influential reference
• Bacillus subtilis antibiotics: structures, syntheses and specific functions

  2005influential reference
• The evolving role of natural products in drug discovery

  2005cited by this paper
• Investigation of the electrophysiological properties of enniatins.

  2004cited by this paper
• Efficacy of fusafungine in acute rhinopharyngitis: a pooled analysis.

  2004cited by this paper
• New shuttle vectors for ectopic insertion of genes into Bacillus subtilis.

  2004cited by this paper
• Actions of the antibiotic mycotoxins beauvericin and enniatin on mammalian tissue.

  2003cited by this paper
• Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases

  2002cited by this paper
• Enniatin synthetase is a monomer with extended structure: evidence for an intramolecular reaction mechanism

  2002cited by this paper
• Regulation of the Acetoin Catabolic Pathway Is Controlled by Sigma L in Bacillus subtilis

  2001cited by this paper
• Directed biosynthesis of new enniatins.

  2001cited by this paper
• Engineered Biosynthesis of the Peptide Antibiotic Bacitracin in the Surrogate Host Bacillus subtilis *

  2001influential reference
• Biosynthesis of Polyketides in Heterologous Hosts

  2001cited by this paper
• Endotoxin removal from protein solutions.

  2000cited by this paper
• An Operon for a Putative ATP-Binding Cassette Transport System Involved in Acetoin Utilization ofBacillus subtilis

  2000cited by this paper
• Ecs, an ABC transporter of Bacillus subtilis: dual signal transduction functions affecting expression of secreted proteins as well as their secretion

  1999cited by this paper
• A new logic for DNA engineering using recombination in Escherichia coli

  1998cited by this paper
• Expression of aggregation-prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems.

  1997cited by this paper
• Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis.

  1996cited by this paper
• Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis

  1996cited by this paper
• Synthesis of a radiolabeled enniatin cyclodepsipeptide [3H‐methyl]JES 1798

  1996cited by this paper
• A xylose-inducible Bacillus subtilis integration vector and its application.

  1996cited by this paper
• Bacillaene, a novel inhibitor of procaryotic protein synthesis produced by Bacillus subtilis: production, taxonomy, isolation, physico-chemical characterization and biological activity.

  1995cited by this paper
• Arrangement of catalytic sites in the multifunctional enzyme enniatin synthetase.

  1995cited by this paper
• A highly specific D-hydroxyisovalerate dehydrogenase from the enniatin producer Fusarium sambucinum.

  1992influential reference
• On the safety of Bacillus subtilis and B. amyloliquefaciens: a review

  1991cited by this paper
• Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis.

  1991cited by this paper
• srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis

  1991cited by this paper
• Effect of fusafungine on adherence of Haemophilus influenzae type b to human epithelial cells in vitro.

  1991cited by this paper
• Nonribosomal biosynthesis of peptide antibiotics.

  1990cited by this paper
• Production of Soluble Recombinant Proteins in Bacteria

  1989cited by this paper
• Constitutive Expression of Enniatin Synthetase during Fermentative Growth of Fusarium scirpi

  1988cited by this paper
• Bactericidal activity of fusafungine towards nosocomial methicillin-resistant staphylococcal strains.

  1988cited by this paper
• Comparative Analysis

  1987cited by this paper
• Enniatin synthetase, a novel type of multifunctional enzyme catalyzing depsipeptide synthesis in Fusarium oxysporum.

  1982cited by this paper
• Growth inhibition of Escherichia coli K-12 by L-valine: A consequence of a regulatory pattern

  1977cited by this paper
• Branched-chain amino acid catabolism in bacteria.

  1976cited by this paper
• Acetoin degradation in Bacillus subtilis by direct oxidative cleavage.

  1975cited by this paper
• Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation.

  1968cited by this paper
• Isoleucine and valine metabolism in Escherichia coli. XI. Valine inhibition of the growth of Escherichia coli strain K-12.

  1962cited by this paper
• ISOLEUCINE AND VALINE METABOLISM IN ESCHERICHIA COLI XI. K-12

  1962cited by this paper

• Engineering non-ribosomal peptide synthesis: tuning the antibiotics engine of the microbial world.

  2026cites this paper
• Development of microbial chassis for production of fungal natural products

  2026cites this paper
• Exploration and Confirmation of the Indole-3-Acetic Acid Biosynthetic Pathway in Plant Growth-Promoting Rhizobacteria Priestia megaterium W101.

  2026cites this paper
• Engineering bacterial cell morphology for the design of robust cell factories

  2025cites this paper
• High-efficiency heterologous expression of functional RVG29-BoNT/A light chain fusion protein in Bacillus subtilis based on a combinatorial strategy.

  2025cites this paper
• A new paradigm for the regulation of A40926B0 biosynthesis

  2025cites this paper
• Highly efficient genome editing in Bacillus subtilis via miniature DNA nucleases IscB

  2025cites this paper
• Response surface methodology to optimize the Bacillussubtilis var. sojaesemen praeparatum liquid fermentation process for the production of fibrinolytic enzyme

  2024cites this paper
• Bacillus subtilis as a host for natural product discovery and engineering of biosynthetic gene clusters.

  2024cites this paper
• Molecular modification and biotechnological applications of microbial aspartic proteases

  2023cites this paper
• Recent advances on cyclodepsipeptides: biologically active compounds for drug research

  2023cites this paper
• Antimicrobial Peptides: Sources, Expression Systems, and Applications.

  2023cites this paper
• Hydroxy Acid Activation in Fungal Non‐Ribosomal Peptide Synthesis Assessed by Site‐Directed Mutagenesis

  2023cites this paper
• Efficient Large-Scale and Scarless Genome Engineering Enables the Construction and Screening of Bacillus subtilis Biofuel Overproducers

  2022cites this paper
• An efficient CRISPR/Cas9‐based genome editing system for alkaliphilic Bacillus sp. N16‐5 and application in engineering xylose utilization for d‐lactic acid production

  2022cites this paper
• A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis

  2022cites this paper
• Development of an efficient iterative genome editing method in Bacillus subtilis using the CRISPR‐AsCpf1 system

  2022cites this paper
• Transcriptome Analysis of Bacillus licheniformis for Improving Bacitracin Production.

  2022cites this paper
• Microbial production of small peptide: pathway engineering and synthetic biology

  2021cites this paper
• Biotechnological Strategies of Riboflavin Biosynthesis in Microbes

  2021cites this paper
• Recent Advances in the Heterologous Biosynthesis of Natural Products from Streptomyces

  2021cites this paper
• Current State and Future Directions of Genetics and Genomics of Endophytic Fungi for Bioprospecting Efforts

  2021cites this paper
• Engineering Heterologous Hosts for the Enhanced Production of Non-ribosomal Peptides

  2020cites this paper
• General Strategies for Biosynthetic Gene Cluster Identification, Capture, and Heterologous Expression

  2020cites this paper
• Single-gene regulated non-spore-forming Bacillus subtilis: Construction, transcriptome responses and applications for producing enzymes and surfactin.

  2020cites this paper
• Design and Construction of Portable CRISPR-Cpf1-Mediated Genome Editing in Bacillus subtilis 168 Oriented Toward Multiple Utilities

  2020cites this paper
• Strategies for recombinant production of antimicrobial peptides with pharmacological potential

  2020cites this paper
• Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites

  2019cites this paper
• Disruption of Protease Genes in Microbes for Production of Heterologous Proteins

  2019cites this paper
• Genetic platforms for heterologous expression of microbial natural products.

  2019cites this paper
• In silico analysis of signal peptides for secretory production of a-amylase in Bacillus subtilis

  2019cites this paper
• Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering.

  2019cites this paper
• Extracellular production of active-form Streptomyces mobaraensis transglutaminase in Bacillus subtilis

  2019cites this paper
• Recent advances in CRISPR/Cas9 mediated genome editing in Bacillus subtilis

  2018cites this paper
• Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon.

  2018cites this paper
• Bacillus subtilis highly efficient protein expression systems that are chromosomally integrated and controllable by glucose and rhamnose

  2018cites this paper
• Recent advances in reconstructing microbial secondary metabolites biosynthesis in Aspergillus spp.

  2018cites this paper
• Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides

  2018cites this paper
• CopySwitch—in vivo Optimization of Gene Copy Numbers for Heterologous Gene Expression in Bacillus subtilis

  2018cites this paper
• A Highly Efficient CRISPR-Cas9-Mediated Large Genomic Deletion in Bacillus subtilis

  2017cites this paper
• Analysis of cellular heterogeneity for enhancing the protein production in industrial scales

  2017cites this paper
• Expression Platforms for Functional Metagenomics: Emerging Technology Options Beyond Escherichia coli

  2017cites this paper
• Staphylococcal serine protease–like proteins are pacemakers of allergic airway reactions to Staphylococcus aureus

  2017cites this paper
• Engineered polyketides: Synergy between protein and host level engineering

  2017cites this paper
• Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis

  2017cites this paper
• Inducible expression of trehalose synthase in Bacillus licheniformis.

  2017cites this paper
• Construction of an enzymatic route using a food-grade recombinant Bacillus subtilis for the production and purification of epilactose from lactose.

  2017cites this paper
• Recent advances in engineering nonribosomal peptide assembly lines.

  2016cites this paper
• A novel strategy for acetonitrile wastewater treatment by using a recombinant bacterium with biofilm-forming and nitrile-degrading capability.

  2016cites this paper
• Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering

  2016cites this paper
• Biodegradation of endocrine disrupting dibutyl phthalate by a bacterial consortium expressing Sphingobium sp. SM42 esterase

  2016cites this paper
• Rational biosynthetic approaches for the production of new-to-nature compounds in fungi.

  2016cites this paper
• Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

  2015cites this paper
• Scale-up bioprocess development for production of the antibiotic valinomycin in Escherichia coli based on consistent fed-batch cultivations

  2015cites this paper
• Production of the polyketide 6-deoxyerythronolide B in the heterologous host Bacillus subtilis

  2015cites this paper
• Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis.

  2015cites this paper
• Recombinant secretory expression, purification and antimicrobial activity of PR39 in Bacillus subtilis using a maltose-inducible vector

  2015cites this paper