Browsing by Person "Hiller, Eric"

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Fed-batch bioreactor cultivation of Bacillus subtilis using vegetable juice as an alternative carbon source for lipopeptides production: a shift towards a circular bioeconomy
(2024) Gugel, Irene; Vahidinasab, Maliheh; Benatto Perino, Elvio Henrique; Hiller, Eric; Marchetti, Filippo; Costa, Stefania; Pfannstiel, Jens; Konnerth, Philipp; Vertuani, Silvia; Manfredini, Stefano; Hausmann, Rudolf; Gudiña, Eduardo
In a scenario of increasing alarm about food waste due to rapid urbanization, population growth and lifestyle changes, this study aims to explore the valorization of waste from the retail sector as potential substrates for the biotechnological production of biosurfactants. With a perspective of increasingly contributing to the realization of the circular bioeconomy, a vegetable juice, derived from unsold fruits and vegetables, as a carbon source was used to produce lipopeptides such as surfactin and fengycin. The results from the shake flask cultivations revealed that different concentrations of vegetable juice could effectively serve as carbon sources and that the fed-batch bioreactor cultivation strategy allowed the yields of lipopeptides to be significantly increased. In particular, the product/substrate yield of 0.09 g/g for surfactin and 0.85 mg/g for fengycin was obtained with maximum concentrations of 2.77 g/L and 27.53 mg/L after 16 h, respectively. To conclude, this study provides the successful fed-batch cultivation of B. subtilis using waste product as the carbon source to produce secondary metabolites. Therefore, the consumption of agricultural product wastes might be a promising source for producing valuable metabolites which have promising application potential to be used in several fields of biological controls of fungal diseases.
Genetic code expansion for controlled surfactin production in a high cell-density Bacillus subtilis strain
(2025) Hermann, Alexander; Hiller, Eric; Hubel, Philipp; Biermann, Lennart; Benatto Perino, Elvio Henrique; Kuipers, Oscar Paul; Hausmann, Rudolf; Lilge, Lars; Fouillaud, Mireille
Background: In biotechnology, B. subtilis is established for heterologous protein production. In addition, the species provides a variety of bioactive metabolites, including the non-ribosomally produced surfactin lipopeptide. However, to control the formation of the target product-forming enzyme, different expression systems could be introduced, including the principle of genetic code expansion by the incorporation of externally supplied non-canonical amino acids. Methods: Integration of an amber stop codon into the srfA operon and additional chromosomal integration of an aminoacyl-tRNA synthetase/tRNA mutant pair from Methanococcus jannaschii enabled site-directed incorporation of the non-canonical amino acid O-methyl-L-tyrosine (OMeY). In different fed-batch bioreactor approaches, OMeY-associated surfactin production was quantified by high-performance thin-layer chromatography (HPTLC). Physiological adaptations of the B. subtilis production strain were analyzed by mass spectrometric proteomics. Results: Using a surfactin-forming B. subtilis production strain, which enables high cell density fermentation processes, the principle of genetic code expansion was introduced. Accordingly, the biosynthesis of the surfactin-forming non-ribosomal peptide synthetase (NRPS) was linked to the addition of the non-canonical amino acid OMeY. In OMeY-associated fed-batch bioreactor fermentation processes, a maximum surfactin titre of 10.8 g/L was achieved. In addition, the effect of surfactin induction was investigated by mass spectrometric proteome analyses. Among other things, adaptations in the B. subtilis motility towards a more sessile state and increased abundances of surfactin precursor-producing enzymes were detected. Conclusions: The principle of genetic code expansion enabled a precise control of the surfactin bioproduction as a representative of bioactive secondary metabolites in B. subtilis . This allowed the establishment of inducer-associated regulation at the post-transcriptional level with simultaneous use of the native promoter system. In this way, inductor-dependent control of the production of the target metabolite-forming enzyme could be achieved.
Glucoselipid biosurfactant biosynthesis operon of Rouxiella badensis DSM 100043T: screening, identification, and heterologous expression in Escherichia coli
(2025) Harahap, Andre Fahriz Perdana; Treinen, Chantal; Van Zyl, Leonardo Joaquim; Williams, Wesley Trevor; Conrad, Jürgen; Pfannstiel, Jens; Klaiber, Iris; Grether, Jakob; Hiller, Eric; Vahidinasab, Maliheh; Perino, Elvio Henrique Benatto; Lilge, Lars; Burger, Anita; Trindade, Marla; Hausmann, Rudolf; Seo, Myung-Ji
Rouxiella badensis DSM 100043T had been previously proven to produce a novel glucoselipid biosurfactant which has a very low critical micelle concentration (CMC) as well as very good stability against a wide range of pH, temperature, and salinity. In this study, we performed a function-based library screening from a R. badensis DSM 100043T genome library to identify responsible genes for biosynthesis of this glucoselipid. The identified open reading frames (ORFs) were cloned into several constructs in Escherichia coli for gene permutation analysis and the individual products were analyzed using high-performance thin-layer chromatography (HPTLC). Products of interest from positive expression strains were purified and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) for further structure elucidation. Function-based screening of 5400 clones led to the identification of an operon containing three ORFs encoding acetyltransferase GlcA (ORF1), acyltransferase GlcB (ORF2), and phosphatase/HAD GlcC (ORF3). E. coli pCAT2, with all three ORFs, resulted in the production of identical R. badensis DSM 100043T glucosedilipid with Glu-C10:0-C12:1 as the main congener. ORF2-deletion strain E. coli pAFP1 primarily produced glucosemonolipids, with Glu-C10:0,3OH and Glu-C12:0 as the major congeners, predominantly esterified at the C-2 position of the glucose moiety. Furthermore, fed-batch bioreactor cultivation of E. coli pCAT2 using glucose as the carbon source yielded a maximum glucosedilipid titer of 2.34 g/L after 25 h of fermentation, which is 55-fold higher than that produced by batch cultivation of R. badensis DSM 100043T in the previous study.
The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes
(2024) Hiller, Eric; Off, Manuel; Hermann, Alexander; Vahidinasab, Maliheh; Benatto Perino, Elvio Henrique; Lilge, Lars; Hausmann, Rudolf
Background The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled. Results Using different growth rates in the range of 0.075 and 0.4 h-1, highest surfactin titres of 36 g/L were reached at 0.25 h-1 with production yields YP/S of 0.21 g/g and YP/X of 0.7 g/g, while growth rates lower than 0.2 h-1 resulted in insufficient and slowed biomass formation as well as surfactin production (YP/S of 0.11 g/g and YP/X of 0.47 g/g for 0.075 h-1). In contrast, feeding rates higher than 0.25 h-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1. Conclusions Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates.
Structure elucidation and characterization of novel glycolipid biosurfactant produced by Rouxiella badensis DSM 100043T
(2025) Harahap, Andre Fahriz Perdana; Conrad, Jürgen; Wolf, Mario; Pfannstiel, Jens; Klaiber, Iris; Grether, Jakob; Hiller, Eric; Vahidinasab, Maliheh; Salminen, Hanna; Treinen, Chantal; Perino, Elvio Henrique Benatto; Hausmann, Rudolf; Serianni, Anthony S.
Microbial biosurfactants have become increasingly attractive as promising ingredients for environmentally friendly products. The reasons for this are their generally good performance and biodegradability, low toxicity, production from renewable raw materials, and benefits for the environment perceived by consumers. In this study, we investigated the chemical structure and properties of a novel glycolipid from a new biosurfactant-producing strain, Rouxiella badensis DSM 100043 T . Bioreactor cultivation was performed at 30 °C and pH 7.0 for 28 h using 15 g/L glycerol as a carbon source. The glycolipid was successfully purified from the ethyl acetate extract of the supernatant using medium pressure liquid chromatography (MPLC). The structure of the glycolipid was determined by one- and two-dimensional ( 1 H and 13 C) nuclear magnetic resonance (NMR) and confirmed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). NMR analysis revealed the hydrophilic moiety as a glucose molecule and the hydrophobic moieties as 3-hydroxy-5-dodecenoic acid and 3-hydroxydecanoic acid, which are linked with the glucose by ester bonds at the C2 and C3 positions. Surface tension measurement with tensiometry indicated that the glucose–lipid could reduce the surface tension of water from 72.05 mN/m to 24.59 mN/m at 25 °C with a very low critical micelle concentration (CMC) of 5.69 mg/L. Moreover, the glucose–lipid demonstrated very good stability in maintaining emulsification activity at pH 2–8, a temperature of up to 100 °C, and a NaCl concentration of up to 15%. These results show that R. badensis DSM 100043 T produced a novel glycolipid biosurfactant with excellent surface-active properties, making it promising for further research or industrial applications.