Stefan Junne

Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic Clostridium acetobutylicum Fermentations

Plasmid pAADB1 for the overexpression of the alcohol-aldehyde dehydrogenase (aad) gene and downregulation of the coenzyme A transferase (CoAT) using antisense RNA (asRNA) against ctfB (the second CoAT gene on the polycistronic aad-ctfA-ctfB message) was used in order to increase the butanol/acetone ratio of Clostridium acetobutylicum ATCC 824 fermentations. Acetone and butanol levels were drastically reduced in 824(pCTFB1AS) (expresses only an asRNA against ctfB) compared to 824(pSOS95del) (plasmid control). Compared to strain 824(pCTFB1AS), 824(pAADB1) fermentations exhibited two profound differences. First, butanol levels were ca. 2.8-fold higher in 824(pAADB1) and restored back to plasmid control levels, thus supporting the hypothesis that asRNA downregulation of ctfB leads to degradation of the whole aad-ctfA-ctfB transcript. Second, ethanol titers in 824(pAADB1) were ca. 23-fold higher and the highest (ca. 200 mM) ever reported in C. acetobutylicum. Western blot analysis confirmed that CoAT was downregulated in 824(pAADB1) at nearly the same levels as in strain 824(pCTFB1AS). Butyrate depletion in 824(pAADB1) fermentations suggested that butyryl-CoA was limiting butanol production in 824(pAADB1). This was confirmed by exogenously adding butyric acid to 824(pAADB1) fermentations to increase the butanol/ethanol ratio. DNA microarray analysis showed that aad overexpression profoundly affects the large-scale transcriptional program of the cells. Several classes of genes were differentially expressed [strain 824(pAADB1) versus strain 824(pCTFB1AS)], including genes of the stress response, sporulation, and chemotaxis. The expression patterns of the CoAT genes (ctfA and ctfB) and aad were consistent with the overexpression of aad and asRNA downregulation of ctfB.

Scale-down simulators for metabolic analysis of large-scale bioprocesses.

Analytical approaches for a comprehensive understanding of the metabolic networks in microbial cultures are mostly based on small-scale cultures which are in a steady state or undergo dynamic changes. For drawing conclusions to industrial-scale bioprocesses, however, it is important to understand that cells in large-scale bioreactors are exposed steadily to fast changes, because of an inhomogeneous environment. Analytical approaches that aim for large-scale bioprocess understanding need to apply specific laboratory simulators. Recent developments in cell cultivation techniques and computational tools provide improved possibilities to evaluate how a process will behave in the final scale. These simulators will pave the way for screening robust strains and process conditions.

High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor

BackgroundSingle-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production.ResultsCultivation of E. coli in the BIOSTAT® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD600) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD600 of 60 (20 g L-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase® Flo, OD600 of 30 (10 g L-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor.ConclusionsRocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

Consistent development of bioprocesses from microliter cultures to the industrial scale

Bioprocess development today is slow and expensive compared to chemical process development. A drastic paradigm shift is necessary and possible by the consistent application of engineering strategies that are typically used in the process development phase already in the early product development. Aside from providing a consistent pathway, strategies such as statistical‐based design of experiments, fed‐batch, minibioreactors, new on‐line sensors, process modeling, and control tools in combination with automation of manual steps offer a higher success rate and the opportunity to find the optimum parameters and operation point. This also directly benefits the early phases of biomolecular screening and initial production of small amounts of the target molecule. The paper reviews the bioprocess developmental phases from a business perspective and the available systems and technologies.

A two‐compartment bioreactor system made of commercial parts for bioprocess scale‐down studies: Impact of oscillations on Bacillus subtilis fed‐batch cultivations

This study describes an advanced version of a two-compartment scale-down bioreactor that simulates inhomogeneities present in large-scale industrial bioreactors on the laboratory scale. The system is made of commercially available parts and is suitable for sterilization with steam. The scale-down bioreactor consists of a usual stirred tank bioreactor (STR) and a plug flow reactor (PFR) equipped with static mixer modules. The PFR module with a working volume of 1.2 L is equipped with five sample ports, and pH and dissolved oxygen (DO) sensors. The concept was applied using the non-sporulating Bacillus subtilis mutant strain AS3, characterized by a SpoIIGA gene knockout. In a fed-batch process with a constant feed rate, it is found that oscillating substrate and DO concentration led to diminished glucose uptake, ethanol formation and an altered amino acid synthesis. Sampling at the PFR module allowed the detection of dynamics at different concentrations of intermediates, such as pyruvic acid, lactic acid and amino acids. Results indicate that the carbon flux at excess glucose and low DO concentrations is shifted towards ethanol formation. As a result, the reduced carbon flux entering the tricarboxylic acid cycle is not sufficient to support amino acid synthesis following the oxaloacetic acid branch point.

Response of Corynebacterium glutamicum exposed to oscillating cultivation conditions in a two‐ and a novel three‐compartment scale‐down bioreactor

The oscillatory conditions in substrate and oxygen supply that typically occur on a large (industrial) scale are usually simulated in two‐compartment scale‐down reactors. In this study, the performance of nutrient‐limited fed‐batch cultivations of Corynebacterium glutamicum in a standard two‐compartment reactor (two‐CR) is compared to the performance in a novel three‐compartment reactor (three‐CR). The three‐CR is designed to mimic three distinct zones of an industrial scale bioreactor that occur if the feed addition is installed at the bottom of the fluid phase. Our findings show that lactate and succinate appear in concentrations two‐fold higher in the three‐CR cultivation than in the two‐CR cultivation. Similar results are revealed for the amino acids glycine, threonine, glutamate, and glutamine. In contrast to the two‐CR cultivation, no intracellular accumulation of pyruvate is observed in the three‐CR cultivation, since the carbon fluxes are directed toward lactate. As previously reported, the expression of lactate dehydrogenase (LDH) is increased in the context of oxygen deprivation. Thus, C. glutamicum adapts to the oscillating environment in the three‐CR. This successful adaptation is revealed by a flow cytometric analysis of BOX‐stained cells and a series of electrooptical at line measurements of cell polarisability. Both methods indicate a higher polarisability of cells in the three‐CR cultivation. PI‐staining does not indicate any membrane damage or accelerated cell death in either system. However, although the strain shows robustness, the product yield of lysine is reduced in scale‐down cultivations as compared to cultivations at homogeneous conditions, which underlines the relevance of process optimization. Biotechnol. Bioeng. 2015;112: 1220–1231.

Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system

The enzyme controlled substrate delivery cultivation technology EnBase(®) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.

Assessment of robustness against dissolved oxygen/substrate oscillations for C. glutamicum DM1933 in two-compartment bioreactor.

Corynebacterium glutamicum is an important organism for industrial biotechnology; particularly, in amino acid production (e.g. l-lysine). Production scales often reach reactor working volumes of several hundred cubic meters, which triggers inhomogeneous distribution of substrates and dissolved gasses due to increasing mixing times. Individual cells which follow the flow profile through the reactor are experiencing oscillating microenvironments. Oscillations can have an influence on the process performance, which is a subject of scale-down experiments. In this work, l-lysine-producing C. glutamicum DM1933 was assessed for its robustness against continuous dissolved oxygen and substrate supply oscillation in two-compartment scale-down bioreactors. Aerobic, substrate-limited stirred tank and non-aerated, substrate-excess plug flow compartments were applied for oscillation. Inhomogeneity of substrate and oxygen supply was observed to cause rapid side product turnover, redistribution of oxygen uptake from oxygen limited into fully aerobic zones, and intermediate medium acidification. However, process inhomogeneity did not impair productivity or growth at plug flow residence times of several minutes. In a focused analysis of proteome, metabolome, transcriptome, and other physiological parameters, no changes were identified in response to process inhomogeneity. In conclusion, fed-batch processes with C. glutamicum DM1933 possess remarkable robustness against oxygen and substrate supply oscillation, which is a unique property in the field of published scale-down studies. Microbial physiology of C.glutamicum appears to be ideally adapted to both homogeneous and inhomogeneous conditions. This ensures exceptional suitability for cultivation at increased mixing times, which is suggested to constitute an important basis for the long-lasting success in large scale bioprocess application.

Cultivation of Marine Microorganisms in Single-Use Systems

: Marine cultures are an important source of novel substances and enzymes. As efforts to isolate strains from (deep) sea environments increase, the demand for methodology platforms to cultivate these organisms is also rising. Due to the high salt concentration and the shear sensitivity exhibited by some heterotrophic microalgae, single-use systems originally designed for the cultivation of mammalian cell lines can be a valuable alternative. Using the cultivation of the heterotrophic marine microalgae Crypthecodinium cohnii as an example, this chapter makes suggestions for experimental design, for improving process development by integrating parallel experiments, and for scaling-up and scaling-down methodologies. It describes how to identify suitable single-use systems and how to integrate a two-layer system with perfluordecalin to improve the gas transfer in deep-well plates. The process is also scaled up in several single-use systems. We also describe challenges in the process development to achieve sufficient oxygen transfer, monitoring, and control, and we discuss limitations such as corrosion, long-term stability, and leachables in single-use systems. Finally, we demonstrate a method for cheap, fast, and consistent process development for marine microorganisms.

Growth and docosahexaenoic acid production performance of the heterotrophic marine microalgae Crypthecodinium cohnii in the wave‐mixed single‐use reactor CELL‐tainer

The process development of docosahexaenoic acid (DHA) with the heterotrophic marine microalgae Crypthecodinium cohnii is challenging, since the oxygen demand of the cells is high and the cell itself sensitive to high shear forces. Furthermore, the marine medium, which comprises a high salt concentration, accelerates corrosion of steel typically applied in bioreactors. Single‐use bioreactors (SUBs) provide an alternative to common stainless steel stirred tank reactors (STR) for the development of bioprocesses with marine microorganisms, when the development stage does not allow for the application of special steel or the replacement of chloride ions in the medium. Among the readily‐available SUBs, the CELL‐tainer exhibits outstanding high oxygen transfer rates. A direct comparison of this SUB with a standard stirred tank bioreactor (STR) was performed for the cultivation of C. cohnii. The biomass and DHA concentrations were similar in both devices, while the biomass yield coefficient, related to the glucose consumption, was reduced by 35% and the DHA yield coefficient by 21% in the STR. The impact of shear stress on cell physiology was elucidated by flow cytometry, revealing a five times higher amount of cell debris in the STR and 14 times higher amount of cells with a low cell membrane potential as evaluated by bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol staining. Foam and biofilm formation, which is a problem in the STR, did not occur in the SUB. The study underlines that the CELL‐tainer can replace the STR, ensuring sufficient oxygen transfer rates and decreasing cell damage and foam formation.