Browsing by Subject "Biotensid"

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Applied molecular bioprocess control using RNA thermometers : exploiting temperature responsive elements for rhamnolipid production
(2022) Noll, Philipp; Hausmann, Rudolf
The highest titer reported for heterologous Rhamnolipid (RL) production is 14.9 g/L. However, biomass generation, as a large carbon sink, was a significant drawback in this process with roughly 50 more biomass than product produced. This problem is addressed in this thesis leveraging temperature as control variable and a molecular temperature sensor, an RNA thermometer (RNAT). RNAT generally refers to secondary loop structures, in the 5’ untranslated region of the mRNA, that form at certain temperatures and therefore regulate translation in dependence of temperature. The ROSE (repression of heat shock gene expression) RNAT evaluated in the first original research article in the heterologous system P. putida KT2440 pSynpro8oT_rhlAB originates from P. aeruginosa. The ROSE element regulates, in dependence of ambient temperature, the translation of rhlA and via a polar effect also the translation of rhlB therefore indirectly RL synthesis. It was found that in the ROSE RNAT-controlled system, the RL production rate was 60% higher at cultivations of 37°C than at 30°C. However, besides the regulatory effect of the RNAT, as revealed by control experiments, multiple unspecific metabolic effects may be equally responsible for the increase in production rate. After screening for even more efficient regulatory structures, a fourU RNAT was identified. Natively, this fourU RNAT regulates the expression of the heat shock gene agsA of Salmonella enterica and its regulatory capability can easily be modified by site-directed mutagenesis. The experimental data collected in the second original research article confirms the functionality of the fourU RNAT in the heterologous RL production system. The data suggested improved regulatory capabilities of the fourU RNAT compared to the ROSE element and a major effect of temperature on RL production rates and yields. The average RL production rate increased by a factor of 11 between 25°C and 38°C. Control experiments confirmed that a major part of this increase originates from the regulatory effect of the fourU RNAT rather than from an unspecific metabolic effect. With this system YP/X values well above 1 (about 1.4 gRL/gBM) could be achieved mitigating the problem of high biomass formation compared to product synthesis. Also, YP/S values of about 0.2 gRL/gGlc at elevated temperatures of 37-38°C were reached in shake flasks. The system was subsequently tested in a proof-of-concept bioreactor process involving a temperature switch. With this simple batch experiment and a temperature switch from 25°C to 38°C not only a partial decoupling of biomass formation from product synthesis was achieved but also an around 25% higher average specific rhamnolipid production rate reached compared to the so far best performing heterologous RL production process reported in literature (average specific production rate: 24 mg/(g h) vs. 32 mg/(g h)). However, to achieve higher titers while reducing side product formation a suitable feeding strategy and more complex temperature profiles may be required. Temperature variations in turn cause several metabolic changes, many of which are complex and interdependent. Models that describe biological processes as a function of temperature are thus essential for improved process understanding. The goal of the peer reviewed review article “Modeling and Exploiting Microbial Temperature Response”, shown in this thesis, was to present an overview of various temperature models, aid comprehension of model intent and to facilitate selection and application. Since not all metabolic interdependencies and mechanisms during temperature variation are known for the reasonable connection of input-output relationships, a suitable modeling approach seemed to be neural networks. Neural networks as black box models do not require mechanistic a priori knowledge but representative historic datasets. To collect training data, different temperature profiles or constant temperatures for a bioreactor process with P. putida KT2440 pSynpro8oT_rhlAB were applied and concentration curves for biomass, glucose and RL recorded. Subsequently, the data was fed into the neural network to compute RL titer as output. An exponential temperature profile yielded at the highest RL value of approx. 9 g (around 13 g/L) less biomass (around 12 g/L) than product. These values were reached after only 30 h consuming just 45 g of glucose. Hence, at this timepoint 36 weight-% of the consumed glucose could be assigned to mono-RL (YP/S = 0.19 gRL/gGlc) and biomass (YX/S = 0.17 gBM/gGlc. The so far best performing heterologous RL production process, yielded 23.2 g (14.9 g/L) mono-RL from >250 g of consumed glucose (YP/S = 0.10 gRL/gGlc) in >70 h using the same strain and medium but a constant temperature of 30°C.
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.