Browsing by Subject "Lipopeptide"

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Characterization of Bacillus velezensis UTB96, demonstrating improved lipopeptide production compared to the strain B. velezensis FZB42
(2022) Vahidinasab, Maliheh; Adiek, Isabel; Hosseini, Behnoush; Akintayo, Stephen Olusanmi; Abrishamchi, Bahar; Pfannstiel, Jens; Henkel, Marius; Lilge, Lars; Vögele, Ralf ; Hausmann, Rudolf
Bacillus strains can produce various lipopeptides, known for their antifungal properties. This makes them attractive metabolites for applications in agriculture. Therefore, identification of productive wild-type strains is essential for the development of biopesticides. Bacillus velezensis FZB42 is a well-established strain for biocontrol of plant pathogens in agriculture. Here, we characterized an alternative strain, B. velezensis UTB96, that can produce higher amounts of all three major lipopeptide families, namely surfactin, fengycin, and iturin. UTB96 produces iturin A. Furthermore, UTB96 showed superior antifungal activity towards the soybean fungal pathogen Diaporthe longicolla compared to FZB42. Moreover, the additional provision of different amino acids for lipopeptide production in UTB96 was investigated. Lysine and alanine had stimulatory effects on the production of all three lipopeptide families, while supplementation of leucine, valine and isoleucine decreased the lipopeptide bioproduction. Using a 45-litre bioreactor system for upscaling in batch culture, lipopeptide titers of about 140 mg/L surfactin, 620 mg/L iturin A, and 45 mg/L fengycin were achieved. In conclusion, it becomes clear that B. velezensis UTB96 is a promising strain for further research application in the field of agricultural biological controls of fungal diseases.
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.
Exploiting novel strategies for the production of surfactin in Bacillus subtilis cultures
(2021) Hoffmann, Mareen; Hausmann, Rudolf
Biosurfactants are synthesized by various microorganisms. These surface-active molecules are a promising alternative to petrochemically and oleochemically produced surfactants. Advantageously, biosurfactants are reported to be better biodegradable and less toxic. The cyclic lipopeptide surfactin synthesized by Bacillus subtilis displays one interesting biosurfactant. Many studies report on the outstanding physico-chemical characteristics and add on benefits such as antimicrobial properties. Hence, surfactin has the potential to be used in a variety of industrial sectors. Nevertheless, processes ensuring both robustness and high titers are rare, especially as conventional aerobic bioreactor cultivations share one major disadvantage, namely excessive foaming. To approach industrial processes, different methods are applied, which can be categorized in three practices. These are (1) media and process parameter optimization, (2) strain engineering, and (3) investigating novel process strategies. For the latter category, the anaerobic growth by nitrate respiration poses an interesting foam-free alternative. In this sense, the anaerobic cultivation of B. subtilis to produce surfactin coupled with the three afore mentioned practices was addressed in this thesis targeting at a foam-free surfactin production process. In the 1st publication, the genome reduced strain B. subtilis IIG-Bs20-5-1, a derivative of the laboratory strain 168 able to synthesize surfactin, was evaluated with respect to its suitability as surfactin producer at various temperatures under both aerobic and anaerobic conditions. It was hypothesized that a deletion of 10% of the genome, e.g., non-essential genes synthesizing prophages or the antibiotic bacilysin, saves metabolic resources and hence results in increased surfactin titers. Strains B. subtilis JABs24, a 168 derivative able to synthesize surfactin but without genome reduction, and the surfactin producer B. subtilis DSM 10T served for comparison. Although strain IIG-Bs20-5-1 was superior regarding specific growth rate µ and biomass yield YX/S, the strain was inferior with respect to surfactin titers, product related yields YP/S and YP/X, and specific productivity q. Indeed, compared to others in literature described strains, B. subtilis JABs24 was emphasized as promising target strain for further process development, reaching high surfactin titers of 1147 mg/L aerobically and 296 mg/L anaerobically as well as exceptionally high product yields YP/X under anaerobic conditions. Accordingly, iterative process optimization was hypothesized to improve anaerobically achieved surfactin titers. However, several aspects to consider of anaerobic growth of B. subtilis by nitrate respiration were described in the 2nd publication. Amongst others, increasing ammonium concentrations, resulting from nitrate reduction to ammonium via nitrite, were shown to have no impact on growth of strain JABs24, but surfactin titers and expression of nitrate reductase promoter PnarG were reduced. Nitrite was shown to peak within the first hours of cultivation and concentrations up to 10 mmol/L resulted in prolonged lag-phases. Moreover, acetate accumulated drastically during the time course of cultivation independent of glucose availability, thus decreasing the glucose flux into biomass. Acetate additionally influenced both specific growth rate µ and PnarG expression negatively. Concluding, the general feasibility of anaerobic fed-batch cultivations to synthesize surfactin was shown, but several aspects must be addressed in future works to make this strategy an equated process with aerobic cultivations. In the 3rd publication, a self-inducible surfactin synthesis process was presented where expression of the surfactin operon in B. subtilis JABs24 was induced under oxygen limited conditions. The native promoter of the srfA operon PsrfA was replaced by anaerobically inducible nitrate reductase promoter PnarG and nitrite reductase promoter PnasD. Shake flask cultivations with varying oxygen availabilities demonstrated that both PnarG and PnasD can serve as auto-inducible promoters. At high oxygen availability, surfactin was not produced in the promoter exchange strains. At lowest oxygen availability, the strain carrying PnarG reached lower surfactin titers than the native JABs24 strain, although expression levels of PnarG and PsrfA were similar. However, strain B. subtilis MG14 with PsrfA::PnasD reached 1.4-fold higher surfactin titers with 696 mg/L and an exceptionally high YP/X of 1.007 g/g with overall lower foam levels. Though, bioreactor cultivations have illustrated that the anaerobic induction must be performed slowly as to avoid cell lysis, resulting in so-defined aerobic-anaerobic switch processes. With further appropriate process optimization, a simple and robust surfactin production process with highly reduced or even no foam formation can be achieved employing strain B. subtilis MG14.
Influence of B. subtilis 3NA mutations in spo0A and abrB on surfactin production in B. subtilis 168
(2021) Klausmann, Peter; Lilge, Lars; Aschern, Moritz; Hennemann, Katja; Henkel, Marius; Hausmann, Rudolf; Morabbi Heravi, Kambiz
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.