Browsing by Person "Vahidinasab, Maliheh"

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    Characterization ofantifungal properties of lipopeptide-producing Bacillus velezensis strains and their proteome-based response to the phytopathogens, Diaporthe spp
    (2023) Akintayo, Stephen Olusanmi; Hosseini, Behnoush; Vahidinasab, Maliheh; Messmer, Marc; Pfannstiel, Jens; Bertsche, Ute; Hubel, Philipp; Henkel, Marius; Hausmann, Rudolf; Vögele, Ralf; Lilge, Lars
    Introduction: B. velezensis strains are of interest in agricultural applications due to their beneficial interactions with plants, notable through their antimicrobial activity. The biocontrol ability of two new lipopeptides-producing B. velezensis strains ES1-02 and EFSO2-04, against fungal phytopathogens of Diaporthe spp., was evaluated and compared with reference strains QST713 and FZB42. All strains were found to be effective against the plant pathogens, with the new strains showing comparable antifungal activity to QST713 and slightly lower activity than FZB42. Methods: Lipopeptides and their isoforms were identified by high-performance thin-layer chromatography (HPTLC) and mass spectrometric measurements. The associated antifungal influences were determined in direct in vitro antagonistic dual culture assays, and the inhibitory growth effects on Diaporthe spp. as representatives of phytopathogenic fungi were determined. The effects on bacterial physiology of selected B. velezensis strains were analyzed by mass spectrometric proteomic analyses using nano-LC-MS/MS. Results and Discussion: Lipopeptide production analysis revealed that all strains produced surfactin, and one lipopeptide of the iturin family, including bacillomycin L by ES1-02 and EFSO2-04, while QST713 and FZB42 produced iturin A and bacillomycin D, respectively. Fengycin production was however only detected in the reference strains. As a result of co-incubation of strain ES1-02 with the antagonistic phytopathogen D. longicolla, an increase in surfactin production of up to 10-fold was observed, making stress induction due to competitors an attractive strategy for surfactin bioproduction. An associated global proteome analysis showed a more detailed overview about the adaptation and response mechanisms of B. velezensis, including an increased abundance of proteins associated with the biosynthesis of antimicrobial compounds. Furthermore, higher abundance was determined for proteins associated with oxidative, nitrosative, and general stress response. In contrast, proteins involved in phosphate uptake, amino acid transport, and translation were decreased in abundance. Altogether, this study provides new insights into the physiological adaptation of lipopeptide-producing B. velezensis strains, which show the potential for use as biocontrol agents with respect to phytopathogenic fungi.
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    Evaluation and method development for the biosynthesis of microbial lipopeptides by bacillus species
    (2023) Vahidinasab, Maliheh; Hausmann, Rudolf
    Microbial lipopeptides are secondary metabolites produced by bacteria and single-celled microorganisms. They consist of a cyclic or linear peptide chain linked to a lipid residue. Due to their high-foaming biosurfactant properties, they have various industrial applications such as in detergents, food emulsifiers, bioremediation, and enhanced oil recovery. Additionally, they possess other functional properties such as antifungal activity, making them an environmentally friendly alternative to synthetic fertilizers and fungicides. Bacillus species produce cyclic lipopeptides known for their potent antifungal activity, which makes them a potential source of bio-fungicides in agriculture. However, the production titer of wild-type Bacillus species does not meet industrial needs. Thereby, genetic modification of producer strains and bioprocess engineering can help increase the production of lipopeptides. Nevertheless, the regulation and basis of biosynthesis for Bacillus lipopeptides are still not completely understood, and ongoing research aims to enhance their production. In general, three main lipopeptide families, including surfactins, iturins, and fengycins are produced by different Bacillus species. Among these, surfactin as the strong biosurfactant is the most extensively studied lipopeptide produced by Bacillus species. The focus of this doctoral thesis was mainly to evaluate the biosynthesis of iturin and fengycin families, which are strong antimicrobial lipopeptides produced by Bacillus subtilis and Bacillus velezensis. This involved developing strains through genetic engineering and enhancing the lipopeptide titer by evaluating the cultivation medium. Initially, the entire genome of the bacteria used in this thesis was examined in terms of lipopeptide biosynthesis, and the structure and yield of the different produced lipopeptides were analyzed. Regarding the lipopeptide producer derivatives of the domesticated laboratory model strain B. subtilis 168 and B. subtilis 3NA, a spore deficient strain appropriate for bioreactor cultivation, surfactin is the lipopeptide with the highest yield, while plipastatin which is a member of fengycin family, is produced in lower quantities. In the present thesis, the biosynthesis of plipastatin by B. subtilis BMV9 as the lipopeptide producer derivative of strain 3NA was evaluated. The study aimed to convert BMV9 to a constitutive plipastatin mono-producer strain. In this sense, overexpressing plipastatin biosynthesis operon using the stronger constitutive Pveg promoter led to a five-fold increase in plipastatin production. Interestingly, it was observed that deletion of srfAA-AD operon in BMV9 and the constructed constitutive plipastatin producer strain has not improved plipastatin production. Therefore, it can be stated that presumably the biosynthesis of plipastatin may be positively influenced in a post-transcriptional manner by the surfactin synthetase or some of its subunits. However, the regulatory mechanism behind this effect remained unknown and requires further research. Another attempt to enhance the plipastatin biosynthesis in strain BMV9 was repairing the degQ expression. One main genome characterization of strains with B. subtilis 168 and 3NA background is that the pleiotropic degQ gene expression, which is known to have a positive effect on plipastatin biosynthesis, is silenced due to a mutation in the promoter area. However, while repair of degQ expression in BMV9 increased the plipastatin production, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) has not significantly increased the plipastatin yield. To further evaluate the impact of degQ expression on surfactin and plipastatin biosynthesis, two strains of B. subtilis were selected: JABs24, a lipopeptide producer derived from the 168 strain, and DSM10T, the wild-type strain expressing native degQ. The findings demonstrated that surfactin biosynthesis is negatively affected by DegQ-associated DegU regulation, while increased plipastatin biosynthesis is achieved in the presence of native degQ expression. In addition to production of lipopeptides, the DegU regulatory system also plays a role in the formation of secretory proteases. A comparison of extracellular protease activities between JABs24 and DSM10T showed that degQ expression led to DSM10T having five times higher protease activity than JABs24. Interestingly, production of extracellular proteases has not affected the stability of both plipastatin and surfactin during cultivation, suggesting that lipopeptides are less targeted by extracellular proteases. The identification of proficient wild-type strains is critical to the advancement of bio-fungicide in agriculture. Therefore, the subsequent approach of this thesis centered on the production of microbial lipopeptide by wild-type B. velezensis strains. Here, the lipopeptide productivity and antifungal ability of B. velezensis UTB96 was higher than B. velezensis FZB42, as a well-established strain for biocontrol of plant pathogens in agriculture. Furthermore, addition of certain amino acids stimulated lipopeptide production, and using a bioreactor system resulted in enhancement of lipopeptide production, especially iturin A by UTB96. Overall, the doctoral thesis evaluates the biosynthesis of antimicrobial lipopeptides produced by B. subtilis and B. velezensis. The study involves genetic engineering such as promoter exchange, deletion of genes involved in competing biosynthetic pathways and cultivation medium development with amino acid supplementation to enhance the lipopeptide titer. The thesis also identifies B. velezensis UTB96 as a promising candidate for further research to be used as a wild-type antifungal agent in agriculture.
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    Toward effects of hydrophobicity on biosurfactant production by Bacillus subtilis isolates from crude-oil-exposed environments
    (2024) Hashemi, Seyedeh Zahra; Fooladi, Jamshid; Vahidinasab, Maliheh; Hubel, Philipp; Pfannstiel, Jens; Pillai, Evelina; Hrenn, Holger; Hausmann, Rudolf; Lilge, Lars
    Background: Due to their structural features, biosurfactants reveal promising physicochemical properties, making them interesting for various applications in different fields, such as the food, cosmetics, agriculture, and bioremediation sectors. In particular, the bioproduction of surfactin, one of the most potent microbially synthesized biosurfactant molecules, is of great interest. However, since the wild-type productivities are comparably low, stimulatory environmental conditions have to be identified for improved bioproduction This study aims to find a correlation between the hydrophobicity and production of the biosurfactant surfactin by B. subtilis isolates from crude-oil-contaminated soil and water. Methods: The surfactin production yield was characterized in adapted batch cultivations using high-performance thin-layer liquid chromatography (HPTLC). Defined hydrophobic environmental conditions were achieved by supplementation with hexadecane or polystyrene beads, and the effects on biosurfactant production were measured. Adaptations at the protein level were analyzed using mass spectrometry measurements. Results: The correlation between hydrophobicity and surfactin production was characterized using Bacillus subtilis strains ZH1 and P7 isolated from crude-oil-contaminated soil and water. Since these isolates show the biodegradation of crude oil and hexadecane as hydrophobic substrates, respectively, a first-time approach, using polystyrene beads, was applied to provide a hydrophobic environment. Interestingly, contrary to popular opinion, reduced biosurfactant production was determined. Using mass spectrometric approaches, the physiological effects of co-cultivation and the cellular response at the protein level were investigated, resulting in altered quantities of stress proteins and proteins involved in the carbon metabolism counter to polystyrene beads. Conclusions: Contrary to common opinion, increasing hydrophobicity does not have a stimulating effect, and even reduces the effect on the bioproduction of surfactin as the main biosurfactant using selected B. subtilis strains.