Florian David

Getting the big beast to work--systems biotechnology of Bacillus megaterium for novel high-value proteins.

The high industrial relevance of the soil bacterium Bacillus megaterium as host for recombinant proteins is driving systems-wide analyses of its metabolic and regulatory networks. The present review highlights novel systems biology tools available to unravel the various cellular components on the level of metabolic and regulatory networks. These provide a rational platform for systems metabolic engineering of B. megaterium. In line, a number of interesting studies have particularly focused on studying recombinant B. megaterium in its industrial bioprocess environment thus integrating systems metabolic engineering with systems biotechnology and providing the full picture toward optimal processes.

Optimization of antibody fragment production in Bacillus megaterium: the role of metal ions on protein secretion.

The effect of the concentration of metal ions in minimal media has been shown to be very important for the production and secretion of the antibody fragment D1.3 scFv in Bacillus megaterium YYBm1. The best media compositions for biomass and antibody fragment formation were evaluated using a genetic algorithm. The screening was carried out in 96 microtiter deep well plates with 900 μL cultivation volume. In 7 generations, 240 different kinds of media were tested, key elements for production and secretion were detected and a 117% increase in production of antibody fragment compared to the previously used medium could be achieved. In addition, media with a higher biomass formation (+84%) or with both more biomass and a higher production of antibody fragment (Pareto-front members) were found. Interestingly the best media for protein production and secretion were different in their composition, with regards to the metal ion concentration levels. From data derived experimentally and from the genome, magnesium was shown to be one of the key components of the metal ions tested for biomass formation and especially for production and secretion of the antibody fragment D1.3 scFv.

Antibody production in Bacillus megaterium: Strategies and physiological implications of scaling from microtiter plates to industrial bioreactors

Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5-100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a μ-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH(4))(2)SO(4), and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

BackgroundSingle cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.ResultsHere different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied.ConclusionsIt was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

Evaluation of cell damage caused by cold sampling and quenching for metabolome analysis

Cell damage during sampling and quenching for metabolome analysis have been investigated at whole sample level using an OD-based method and ATP loss investigation, and at single cell level by means of flow cytometry. Escherichia coli was cultivated in shake flasks and sampled into several cold quenching solutions during exponential growth phase varying quenching solution composition and sampling temperature. For single cell analysis, the samples were incubated with selective propidium iodide dye and analysed via flow cytometry to differentiate between intact and damaged cells. It was found that every combination of quenching solution, temperature, or cooling rate tested influenced the E. coli cell membrane integrity indicating rupture which will not only let the dye in, but also intracellular ATP out of the cells, which is not desired in in vivo metabolome analysis.

Functionalization of magnetic nanoparticles with high-binding capacity for affinity separation of therapeutic proteins

Magnetic nanoparticles with immobilized metal ligands were prepared for the separation of antibody fragments. First, iron oxide nanoparticles were produced in a solvothermal synthesis using triethylene glycol as solvent and iron(III) acetylacetonate as organic precursor. Via functionalization of the particles with priorly reacted 3-glycidoxypropyltrimethoxysilane and Nα,Nα-bis(carboxymethyl)-l-lysine (NTA), and charging with Ni2+, magnetic affinity adsorbents were obtained. The particles were applied to separate a His-tagged antibody fragment from a heterogeneous protein mixture of a microbial cultivation supernatant. Binding properties and specificity for purification of the target product ABF D1.3 scFv were optimized regarding the GNTA concentration and were found superior as compared to commercially available systems. A molar ratio of 1:2 Fe2O3:GNTA was most beneficial for the specific purification of the antibody fragment.

Discontinuous and continuous purification of single-chain antibody fragments using immobilized metal ion affinity chromatography

This work describes the adsorption-desorption behavior of a histidine-tagged single-chain Fragment variable antibody (D1.3 scFv) on a commercial immobilized metal ion affinity chromatography (IMAC) column. A clarified cell culture supernatant originating from Bacillus megaterium was characterized using single column experiments in a pH-gradient elution mode. The cell culture supernatant containing the antibody fragment D1.3 scFv could be treated in the chromatographic separation process as a pseudo-binary mixture. Adsorption equilibrium constants of the antibody fragment fraction (ABF) and the non-specifically retained protein impurity fraction (IMP) were determined experimentally at constant pH by reinjecting pulses of pooled fractions collected in preliminary batch gradient elution runs. Based on the estimated adsorption equilibrium constants a possible multicolumn open-loop three-zone two-step pH-gradient simulated moving bed (SMB) process is suggested and designed, which possesses the potential to isolate continuously the antibody fragment fraction (ABF) containing the single-chain antibody fragment D1.3 scFv.

Influence of fructose and oxygen gradients on fed-batch recombinant protein production using Bacillus megaterium

Bacillus megaterium expressing a variant of the green fluorescent protein (GFP) was used to investigate the influence of gradients in dissolved oxygen (DO) and fructose concentration on biomass and product formation. For this purpose, several fed‐batch experiments with DO as feed control parameter were performed using suboptimal and optimal adjusted control parameters. In a first approach, suboptimal DO‐feeding was reached by varying randomly the controller output between 10 and 20% of the maximal pumping capacity. This led to fluctuations in DO between 20 and 80% and to substrate gradients around 3 g/L. GFP formation thereby was decreased. In a second approach, an optimal feeding profile was realized using a PI‐control with a constant controller output of 20% was used. Thereby no gradients in substrate concentration, but low‐amplitude and high‐frequency oscillations in the DO concentration were obtained, leading to increased GFP formation. In a final experimental approach, large‐scale conditions were emulated in a two‐compartment scale‐down system consisting of a 3.7‐L stirred tank reactor and a 0.7‐L non‐stirred vessel. DO‐based feeding was applied with the measurement in the 3.7‐L bioreactor and feeding in the small vessel. This setup led to a fairly constant gradient of 20% between both vessels, which also resulted in a decreased GFP productivity.

Membrane fluidity of halophilic ectoine-secreting bacteria related to osmotic and thermal treatment.

In response to sudden decrease in osmotic pressure, halophilic microorganisms secrete their accumulated osmolytes. This specific stress response, combined with physiochemical responses to the altered environment, influence the membrane properties and integrity of cells, with consequent effects on growth and yields in bioprocesses, such as bacterial milking. The aim of this study was to investigate changes in membrane fluidity and integrity induced by environmental stress in ectoine-secreting organisms. The halophilic ectoine-producing strains Alkalibacillus haloalkaliphilus and Chromohalobacter salexigens were treated hypo- and hyper-osmotically at several temperatures. The steady-state anisotropy of fluorescently labeled cells was measured, and membrane integrity assessed by flow cytometry and ectoine distribution. Strong osmotic downshocks slightly increased the fluidity of the bacterial membranes. As the temperature increased, the increasing membrane fluidity encouraged more ectoine release under the same osmotic shock conditions. On the other hand, combined shock treatments increased the number of disintegrated cells. From the ectoine release and membrane integrity measurements under coupled thermal and osmotic shock conditions, we could optimize the secretion conditions for both bacteria.

Ectoine production by Alkalibacillus haloalkaliphilus—Bioprocess development using response surface methodology and model‐driven strategies

Halophile microorganisms are important ectoine producers and are applied in industrial applications. The growth and osmoregulation are distinguished based on the diversity of environmental distribution and require holistic investigations of cultivation condition and osmotic response. The barely investigated halophilic bacterium Alkalibacillus haloalkaliphilus is able to accumulate the compatible solute ectoine, while growing under environmental stress conditions. Since microbial growth and ectoine production are functions of the process key parameters such as salinity, pH value, and temperature, the optimization resulted in three significant process strategies that were estimated by response surface methodology. For each strategy, single‐cell analysis was performed using a newly developed staining method for monitoring the membrane potential of halophilic microorganisms directly in the cultivation medium. The flow cytometric method was shown to reveal the physiological state during ectoine production and has a great potential to be applied for basic research, process optimization, and quality control.