Strategies for improving fengycin production: a review.

Fengycin is an important member of the lipopeptide family with a wide range of applications in the agricultural, food, medical and cosmetic industries. However, its commercial application is severely hindered by low productivity and high cost. Therefore, numerous studies have been devoted to improving the production of fengycin. We summarize these studies in this review with the aim of providing a reference and guidance for future researchers. This review begins with an overview of the synthesis mechanism of fengycin via the non-ribosomal peptide synthetases (NRPS), and then delves into the strategies for improving the fengycin production in recent years. These strategies mainly include fermentation optimization and metabolic engineering, and the metabolic engineering encompasses enhancement of precursor supply, application of regulatory factors, promoter engineering, and application of genome-engineering (genome shuffling and genome-scale metabolic network model). Finally, we conclude this review with a prospect of fengycin production.

iTRAQ-based proteomic analysis of Bacillus subtilis strain NCD-2 regulated by PhoPR two-component system: A comparative analysis with transcriptomics revealed the regulation for fengycin production by branched chain amino acids.

The PhoPR two-component system (TCS) is a signal transduction pathway to regulate the phosphate starvation response in Bacillus subtilis and regulated fengycin production in strain NCD-2 under low phosphate condition. The purpose of this study was to characterize the proteome level responses in the phoP-null mutant (MP) and the phoR-null mutant (MR), and to integrate the proteomics with the transcriptomic data obtained previously. The metabolic pathway for fengycin was predicted based on omics analysis as well as molecular genetics assay. Results showed the proteins and genes associated with biosynthesis of branched chain amino acids (BCAAs) were regulated by PhoPR TCS, and liquid chromatography mass spectrometry (LC-MS) analysis also confirmed that the production of BCAAs was down-regulated in the MP and MR mutants, when compared to wild-type strain NCD-2. Protein network analysis showed that the BCAA metabolism was linked to the biosynthesis of lipopeptides. The MP and MR strains decreased the fengycin production when cultured in modified Landy medium supplied with 0.42 mM phosphate, however, the fengycin production could be restored when the glutamic acid was replaced with BCAAs that were added to modified Landy medium. The lpdV gene, which is responsible for the BCAA degradation process, was deleted in strain NCD-2. Compared with the wild-type strain, the lpdV mutant produced significantly less fengycin in the medium supplied with BCAAs. Considered together, the results of this study indicate that the PhoPR TCS regulates fengycin production by affecting BCAA biosynthesis.

Ribosomally derived lipopeptides containing distinct fatty acyl moieties.

Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non-gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.

Chemoproteomics profiling of surfactin-producing nonribosomal peptide synthetases in living bacterial cells.

Much of our current knowledge on nonribosomal peptide synthetases (NRPSs) is based on studies in which the full NRPS system or each protein domain is expressed in heterologous hosts. Consequently, methods to detect the endogenous activity of NRPSs, under natural cellular conditions, are needed for the study of NRPS cell biology. Here, we describe the in vivo activity-based protein profiling (ABPP) for endogenous NRPSs and its applications to the study of their activities in bacteria. Remarkably, in vitro and in vivo ABPP in the context of the surfactin producer Bacillus subtilis enabled the visualization, tracking, and imaging of an endogenous SrfAB-NRPS with remarkable selectivity and sensitivity. Furthermore, in vivo, ABPP allowed the discovery of the degradation processes of the endogenous SrfAB-NRPS in the context of its native producer bacteria. Overall, this study deepens our understanding of the properties of NRPSs that cannot be addressed by conventional methods.

Gene editing enables rapid engineering of complex antibiotic assembly lines.

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains.

Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B. subtilis strains JABs24 and DSM10T , surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho-transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B. subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14-fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B. subtilis JABs24, a lipopeptide-producing derivative of strain 168, and B. subtilis wild-type strain DSM10T , which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ, a deletion mutant was constructed for DSM10T , while a natively expressed degQ version was integrated into strain JABs24. This allowed strain-specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10T . While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a sixfold increase in plipastatin was observed. In contrast, degQ deletion in DSM10T increased surfactin titer by threefold but decreased plipastatin production by fivefold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

Influence of B. subtilis 3NA mutations in spo0A and abrB on surfactin production in B. subtilis 168.

BACKGROUND: Bacillus subtilis is a well-established host for a variety of bioproduction processes, with much interest focused on the production of biosurfactants such as the cyclic lipopeptide surfactin. Surfactin production is tightly intertwined with quorum sensing and regulatory cell differentiation processes. As previous studies have shown, a non-sporulating B. subtilis strain 3NA encoding a functional sfp locus but mutations in the spo0A and abrB loci, called JABs32, exhibits noticeably increased surfactin production capabilities. In this work, the impacts of introducing JABs32 mutations in the genes spo0A, abrB and abh from 3NA into strain KM1016, a surfactin-forming derivative of B. subtilis 168, was investigated. This study aims to show these mutations are responsible for the surfactin producing performance of strain JABs32 in fed-batch bioreactor cultivations. RESULTS: Single and double mutant strains of B. subtilis KM1016 were constructed encoding gene deletions of spo0A, abrB and homologous abh. Furthermore, an elongated abrB version, called abrB*, as described for JABs32 was integrated. Single and combinatory mutant strains were analysed in respect of growth behaviour, native PsrfA promoter expression and surfactin production. Deletion of spo0A led to increased growth rates with lowered surfactin titers, while deletion or elongation of abrB resulted in lowered growth rates and high surfactin yields, compared to KM1016. The double mutant strains B. subtilis KM1036 and KM1020 encoding Δspo0A abrB* and Δspo0A ΔabrB were compared to reference strain JABs32, with KM1036 exhibiting similar production parameters and impeded cell growth and surfactin production for KM1020. Bioreactor fed-batch cultivations comparing a Δspo0A abrB* mutant of KM1016, KM681, with JABs32 showed a decrease of 32% in surfactin concentration. CONCLUSIONS: The genetic differences of B. subtilis KM1016 and JABs32 give rise to new and improved fermentation methods through high cell density processes. Deletion of the spo0A locus was shown to be the reason for higher biomass concentrations. Only in combination with an elongation of abrB was this strain able to reach high surfactin titers of 18.27 g L-1 in fed-batch cultivations. This work shows, that a B. subtilis strain can be turned into a high cell density surfactin production strain by introduction of two mutations.

A Conserved Nonribosomal Peptide Synthetase in Xenorhabdus bovienii Produces Citrulline-Functionalized Lipopeptides.

The entomopathogenic bacterium Xenorhabdus bovienii exists in a mutualistic relationship with nematodes of the genus Steinernema. Free-living infective juveniles of Steinernema prey on insect larvae and regurgitate X. bovienii within the hemocoel of a host larva. X. bovienii subsequently produces a complex array of specialized metabolites and effector proteins that kill the insect and regulate various aspects of the trilateral symbiosis. While Xenorhabdus species are rich producers of secondary metabolites, many of their biosynthetic gene clusters remain uncharacterized. Here, we describe a nonribosomal peptide synthetase (NRPS) identified through comparative genomics analysis that is widely conserved in Xenorhabdus species. Heterologous expression of this NRPS gene from X. bovienii in E. coli led to the discovery of a family of lipo-tripeptides that chromatographically appear as pairs, containing either a C-terminal carboxylic acid or carboxamide. Coexpression of the NRPS with the leupeptin protease inhibitor pathway enhanced production, facilitating isolation and characterization efforts. The new lipo-tripeptides were also detected in wild-type X. bovienii cultures. These metabolites, termed bovienimides, share an uncommon C-terminal d-citrulline residue. The NRPS lacked a dedicated chain termination domain, resulting in product diversification and release from the assembly line through reactions with ammonia, water, or exogenous alcohols.

Valorization of date juice by the production of lipopeptide biosurfactants by a Bacillus mojavensis BI2 strain: bioprocess optimization by response surface methodology and study of surface activities.

Lipopeptides biosurfactants (BioS) are natural surface-active compounds produced by a variety of microorganisms. They have great interest in environmental, biomedical and agro-industrial fields. However their large-scale application and production is limited by the cost of culture media and the low yield of production. Therefore, the improvement of the production yields and the development of efficient and cost-effective bioprocess became of a great interest. In this aim, we applied the response surface method to optimize an economic BioS production by a newly isolated strain Bacillus mojavensis BI2 on date Juice called "Luegmi" as unique carbon and nitrogen source. Using a Box-Bhenken design, we studied the effect of three independent variables on lipopeptide production; Leugmi concentration, Na2HPO4 and incubation time. The results of this study showed that Leugmi concentration at 25%, Na2HPO4 at 0.1% and incubation time of 24 h were optimal conditions for BioS  production, with a maximum Surface Tension (ST) decreasing capacity of 55% corresponding to 27 mN/m and an Oil Dispersing Activity (ODA) of 30 cm2 corresponding to a diameter of 6 cm. Preliminary characterization of the BioS produced on Luegmi by UV-Spectra and Thin Layer Chromatography showed its lipopeptide nature. Physic-chemical characterization of the produced lipopeptide on Leugmi showed its great surface activities and stabilities at different pH, temperature and salts concentration. The results of this study suggested that Leugmi, an agricultural byproducts can be used as a low-cost substrate to enhance the yield of lipopeptide BioS with great surface activities for potential environmental application.

Residual biomass from surfactin production is a source of arginase and adsorbed surfactin that is useful for environmental remediation.

Lipopeptides are important secondary metabolites produced by microbes. They find applications in environmental decontamination and in the chemical, pharmaceutical and food industries. However, their production is expensive. In the present work we propose three strategies to lower the production costs of surfactin. First, the coproduction of surfactin and arginase in a single growth. Second, extract the fraction of surfactin that adsorbs to the biomass and is removed from the growth medium through centrifugation. Third, use microbial biomass for the remediation of organic and inorganic contaminants. The coproduction of surfactin and arginase was evaluated by factorial design experiments using the LB medium supplemented with arginine. The best conditions for surfactin production were 22 h of growth at 37 °C using LB supplemented with arginine 7.3 g/L. Almost similar conditions were found to produce highest levels of arginase, 24 h and 6.45 g/L arginine. Decontamination of phenol and copper from artificial samples was attained by treatment with residues from lipopeptide production. Thus, cell suspensions and wash-waters used to extract surfactin from the biomass. Cell suspensions were used to successfully remove hydroquinone. Cell suspensions and wash-waters containing surfactin were successfully used to recover copper from solution. Specific monitoring methods were used for phenol and metal solutions, respectively a biosensor based on tyrosinase and either atomic absorption flame ionization spectrometry or absorbance coupled to the Arduino™ platform. Therefore, we report three alternative strategies to lower the production costs in lipopeptide production, which include the effective recovery of copper and phenol from contaminated waters using residues from surfactin production. Sustainable and profitable production of surfactin can be achieved by a coproduction strategy of lipopeptides and enzymes. Lipopeptides are collected in the supernatant and enzymes in the biomass. In addition, lipopeptides that coprecipitate with biomass can be recovered by washing. Lipopeptide wash-waters find applications in remediation and cells can also be used for environmental decontamination.

Genetic engineering of the precursor supply pathway for the overproduction of the nC14-surfactin isoform with promising MEOR applications.

BACKGROUND: Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a ß-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. RESULTS: In this study, the fatty acid precursor supply pathway in Bacillus subtilis 168 for surfactin production was strengthened through two steps. Firstly, pathways competing for the precursors were eliminated with inactivation of pps and pks. Secondly, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed. As a result, the surfactin titer after 24 h of cultivation improved by 34%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC-MS showed that the proportion of nC14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. CONCLUSION: The obtained results identified that enhancing the supply of fatty acid precursor is very essential for the synthesis of surfactin. At the same time, this study also proved that thioesterase BTE can promote the production of nC14-surfactin and experimentally demonstrated its higher surface activity and better performance in oil-washing. These results are of great significance for the MEOR application of surfactin.

Influence of Culture Substrates on Morphology and Function of Pulmonary Alveolar Cells In Vitro.

Cell's microenvironment has been shown to exert influence on cell behavior. In particular, matrix-cell interactions strongly impact cell morphology and function. The purpose of this study was to analyze the influence of different culture substrate materials on phenotype and functional properties of lung epithelial adenocarcinoma (A549) cells. A549 cells were seeded onto two different biocompatible, commercially available substrates: a polyester coverslip (Thermanox™ Coverslips), that was used as cell culture plate control, and a polydimethylsiloxane membrane (PDMS, Elastosil® Film) investigated in this study as alternative material for A549 cells culture. The two substrates influenced cell morphology and the actin cytoskeleton organization. Further, the Yes-associated protein (YAP) and its transcriptional coactivator PDZ-binding motif (TAZ) were translocated to the nucleus in A549 cells cultured on polyester substrate, yet it remained mostly cytosolic in cells on PDMS substrate. By SEM analysis, we observed that cells grown on Elastosil® Film maintained an alveolar Type II cell morphology. Immunofluorescence staining for surfactant-C revealing a high expression of surfactant-C in cells cultured on Elastosil® Film, but not in cells cultured on Thermanox™ Coverslips. A549 cells grown onto Elastosil® Film exhibited morphology and functionality that suggest retainment of alveolar epithelial Type II phenotype, while A549 cells grown onto conventional plastic substrates acquired an alveolar Type I phenotype.

Integrated Omics Strategy Reveals Cyclic Lipopeptides Empedopeptins from Massilia sp. YMA4 and Their Biosynthetic Pathway.

Empedopeptins-eight amino acid cyclic lipopeptides-are calcium-dependent antibiotics that act against Gram-positive bacteria such as Staphylococcus aureus by inhibiting cell wall biosynthesis. However, to date, the biosynthetic mechanism of the empedopeptins has not been well identified. Through comparative genomics and metabolomics analysis, we identified empedopeptin and its new analogs from a marine bacterium, Massilia sp. YMA4. We then unveiled the empedopeptin biosynthetic gene cluster. The core nonribosomal peptide gene null-mutant strains (ΔempC, ΔempD, and ΔempE) could not produce empedopeptin, while dioxygenase gene null-mutant strains (ΔempA and ΔempB) produced several unique empedopeptin analogs. However, the antibiotic activity of ΔempA and ΔempB was significantly reduced compared with the wild-type, demonstrating that the hydroxylated amino acid residues of empedopeptin and its analogs are important to their antibiotic activity. Furthermore, we found seven bacterial strains that could produce empedopeptin-like cyclic lipopeptides using a genome mining approach. In summary, this study demonstrated that an integrated omics strategy can facilitate the discovery of potential bioactive metabolites from microbial sources without further isolation and purification.

Lipopeptides production by a newly Halomonas venusta strain: Characterization and biotechnological properties.

A halotolerant marine strain PHKT of Halomonas venusta was isolated from contaminated seawater as an efficient biosurfactant producer candidate, on low-value substrate (glycerol). The produced biosurfactants (Bios-PHKT) were characterized as lipopeptides molecules, belonging to surfactin and pumilacidin families, by using Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FT-IR) and Tandem Mass Spectrometry (MALDI-TOF/MS-MS). Bios-PHKT has a critical micelle concentration (CMC) of 125 mg/L, and showed a high steadiness against a wide spectrum of salinity (0-120 g/L NaCl), temperature (4-121 °C) and pH (2-12), supporting its powerful tensioactive properties under various environmental conditions. Likewise, the cytotoxic test revealed that the biosurfactant Bios-PHKT, at concentrations lower than 125 µg/mL, was not cytotoxic for human HEK-293 cells since the cell survival is over than 80%. Furthermore, Bios-PHKT lipopeptides showed excellent anti-adhesive and anti-biofilm activities, being able to avoid and disrupt the biofilm formation by certain pathogenic microorganisms. In addition, the biosurfactant Bios-PHKT showed a remarkable anti-proliferative activity towards tumor B16 melanoma cell line. Besides, Bios-PHKT exhibited an excellent in vitro and in vivo wound healing process. In light of these promising findings, Bios-PHKT could be successfully used in different biotechnological applications.

CodY, ComA, DegU and Spo0A controlling lipopeptides biosynthesis in Bacillus amyloliquefaciens fmbJ.

AIM: In the study, we investigated the regulatory effects of these genes (codY, comA, degU and spo0A) on the biosynthesis of three lipopeptides (bacillomycin D, fengycin and surfactin) in Bacillus amyloliquefaciens. METHODS AND RESULTS: The codY, comA, degU and spo0A genes in B. amyloliquefaciens fmbJ were knocked out. The results showed that the productions of bacillomycin D were significantly reduced compared with that of fmbJ. Their deletion induced great changes in the levels of transcripts specifying metabolic pathways, quorum sensing system and substance transport system in fmbJ. Moreover, overexpression of these genes improved the productions of bacillomycin D. In particular, the overexpression of spo0A enhanced bacillomycin D yield up to 648·9 ± 60·9 mg l-1 from 277·3 ± 30·5 mg l-1 . In addition, the yields of surfactin in fmbJΔcodY and fmbJΔdegU were significantly improved, and the regulatory factor CodY had no significant effect on the synthesis of fengycin. CONCLUSIONS: These genes positively regulated the expression of bacillomycin D and fengycin synthase genes in strain fmbJ. However, codY and degU negatively regulated surfactin biosynthesis. Moreover, it was found that CodY had a concentration dependence on bacillomycin D synthesis. Spo0A might play a direct regulatory role in the synthesis and secretion of bacillomycin D. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that genetic engineering of regulatory genes was an effective strategy to improve the yields of antimicrobial lipopeptides and provided promising strains for industrial production of lipopeptides.

The genomic attributes of Cd-resistant, hydrocarbonoclastic Bacillus subtilis SR1 for rhizodegradation of benzo(a)pyrene under co-contaminated conditions.

Bacillus subtilis SR1 is a metal resistant, polyaromatic hydrocarbon-degrading bacterium isolated from petroleum contaminated sites. This study reports the characteristics of the genome of the isolate containing one circular chromosome (4,093,698 bp) annotated into 4155 genes and 4095 proteins. The genome analysis confirmed the presence of multiple catabolic genes: aromatic ring-hydroxylating dioxygenase (COG2146), aromatic ring hydroxylase (COG2368), catechol 2, 3 dioxygenase (COG2514), 4-hydroxybenzoate decarboxylase (COG0043), carboxymuconolactone decarboxylase (COG0599) responsible for the catabolism of aromatic hydrocarbons along with the genes for biosurfactant production and functional genes (czcD and cadA) for resistance to cadmium, zinc, and cobalt. Gas Chromatography-Mass spectroscopy analysis revealed up to 35% in-vitro degradation of benzo(a)pyrene after 21 days of growth along with the production of different intermediate metabolites. The pot trial analysis in the greenhouse condition validated the rhizodegradation of BaP, which was significantly higher in the presence of plant-microbe association (85%) than degradation in bulk soil (68%).

Complete genome sequence of Bacillus velezensis NST6 and comparison with the species belonging to operational group B. amyloliquefaciens.

Bacillus spp. play important roles in production of bioactive natural products with potential agricultural and medical applications. The three families of lipopeptides produced by Bacillus spp. have been most recognized for their antagonistic activity against other microbes, i.e. fengycin, iturin, and surfactin. A novel strain NST6 was isolated from soil and identified as B. velezensis based on phylogenomic analysis. Genome analysis revealed 21 putative biosynthetic gene clusters including the ones responsible for producing bacillomycin and surfactin. However, fengycin cluster was compromised with absence or partial disruption of three non-ribosomal peptide synthetases. Distribution of biosynthetic gene clusters showed that clusters for iturin families were well conserved in 327 genomes of the species belonging to the operational group B. amyloliquefaciens. However, clusters for fengycin and surfactin showed dynamic distribution at gene level. Comparative analysis of closely related species would provide new insights to the diversity in genetic elements for secondary metabolites.

Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production.

BACKGROUND: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis. RESULTS: In this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167. CONCLUSIONS: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

Selection of sponge-associated bacteria with high potential for the production of antibacterial compounds.

The potential of sponge-associated bacteria for the biosynthesis of natural products with antibacterial activity was evaluated. In a preliminary screening 108 of 835 axenic isolates showed antibacterial activity. Active isolates were identified by 16S rRNA gene sequencing and selection of the most promising strains was done in a championship like approach, which can be done in every lab and field station without expensive equipment. In a competition assay, strains that inhibited most of the other strains were selected. In a second round, the strongest competitors from each host sponge competed against each other. To rule out that the best competitors selected in that way represent similar strains with the same metabolic profile, BOX PCR experiments were performed, and extracts of these strains were analysed using metabolic fingerprinting. This proved that the strains are different and have various metabolic profiles, even though belonging to the same genus, i.e. Bacillus. Furthermore, it was shown that co-culture experiments triggered the production of compounds with antibiotic activity, i.e. surfactins and macrolactin A. Since many members of the genus Bacillus possess the genetic equipment for the biosynthesis of these compounds, a potential synergism was analysed, showing synergistic effects between C14-surfactin and macrolactin A against methicillin-resistant Staphylococcus aureus (MRSA).

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis.

BACKGROUND: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. RESULTS: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. CONCLUSIONS: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.