In the old days of industrial fermentation, microbes produced lactic acid, beer, wine, cheese, and other desired products, yet no one knew exactly how the cells functioned.
Now the tools of systems biology are helping to understand, at all levels, what happens inside microbial cells.
“Today, one needs to have a thorough understanding of the cell as a whole. Systems biology provides a quantitative description of the cell and is an important source of new findings to optimize production systems,” according to Ralf Takors, Ph.D., director of the centre of bioprocess engineering at the University of Stuttgart, Germany.
Takors was one of several presenters at “BioSpain”, a conference organized by ASEBIO (Spanish Association of Biotechnology Companies) and held in Bilbao, Spain.
Dr. Takors uses Pseudomonas putida as a model system to illustrate possible applications of systems biology. P. putida has multiple properties that make it ideal for industrial applications. It degrades many types of substrates, its genome is sequenced, and the microbe has a high stress tolerance. These features make it a good model for systems biology studies that provide a holistic picture of metabolic and regulatory interactions.
“If we can understand the basic characteristics and properties of P. putida, we can transfer them to other microbes to improve industrial biotechnological processes,” said Dr. Takors.
In one set of experiments, Dr. Takors focused on butanol as the sole carbon source, which P. putida completely consumes. The energy charge generated was measured in relation to increasing butanol concentrations. Under steady-state conditions, the energy charge stayed at a constant level of 0.85. But when glucose was added, the energy charge fell to 0.4 and coincided with the highest fraction of butanol consumed.
A transcriptome analysis showed that as butanol concentration rose, 40 genes were overexpressed, including ones coding for ADH degradation, ABC efflux exporters, the AgmR cluster, and the tricarboxylic acid cycle (TCA). “These genes indicate a bacterial regulation scenario not expected so far,” said Dr. Takors.
Additionally, a metabolic flux analysis showed that in the presence of both glucose and butanol, their metabolism was decoupled. The TCA cycle totally consumed butanol, whereas glucose solely fueled glycolysis. “By understanding these events we can change systems to include only cells that are high producers of desired end products,” noted Dr. Takors.
Additionally, flow cytometry showed that over time the fraction of cells with single DNA content decreased as did the growth rate. At the same time, the fraction of cells with twice the DNA content and others with multiple copies of DNA increased and replicated. These different types of cell populations “lead to totally different scenarios of growth,” said Dr. Takors, and “likely affect the strain’s production.” He is working on a mechanistic model to explain these new events uncovered by systems biology.
Aqueous Two-Phase Systems
The increasing demand for human biotherapeutics, such as monoclonal antibodies to treat cancer and autoimmune diseases, is expected to double by 2020. “We can produce up to 10 grams per liter of a monoclonal antibody upstream, but downstream processing accounts for 50% of the cost. We need improvements here,” said Raquel Aires-Barros, Ph.D., an associate professor at the Technical University of Lisbon.
Current downstream processing uses centrifugation to remove cells, followed by purification by Protein A chromatography. These methods are expensive and have limitations for the manufacturing of larger biopharmaceuticals.
Aqueous two-phase systems (ATPS) are proving a valuable alternative. ATPS combines high biocompatibility and selectivity with easy and reliable scale up, and it has continuous operation capabilities. “Our goal is to replace Protein A chromatography with ATPS,” explained Dr. Aires-Barros.
In proof-of-concept studies, ATPS successfully isolated IgG, using a microfluidic platform of polyethylene glycol, buffers, and salts. ATPS also was used to isolate and purify CD34+ stem cells directly from whole umbilical cord blood.
“Stem cells are present in very small amounts in cord blood, and we need cost effective methods to isolate and maximize them,” noted Dr. Aires-Barros.
This novel selection method recovered 95% of CD34+ cells with a purifying factor of 245. The results suggest a new way to purify hematopoietic stem cells for a variety of clinical applications. The researchers are scaling up their ATPS process. So far, the team has successfully used ATPS for downstream purification. However, in the future, the technology could replace upstream centrifugation as well.
More efficient bioreactors offer another way to improve bioprocessing. Oscillatory flow reactors (OFR) increase mass transfer rates and reduce oxygen requirements compared to current jacketed stir tank bioreactors. OFR is an example of process intensification, a strategy that strives to dramatically reduce volumes needed to make chemicals and pharmaceuticals.
“We want to do better with less while increasing productivity,” said Jose A. Teixeira, Ph.D., professor of biological engineering at the University of Minho in Braga, Portugal.
The main apparatus for OFR is a tube with periodic constrictions where fluid pulsations (oscillations) are induced by pistons that are installed in the tube inlet or at both ends. Teixeira built a prototype micro-bioreactor based on OFR and used it to produce gamma-decalactone, a peach aroma product.
The time required to obtain the maximum concentration of gamma-decalactone was cut by 50%, compared to a comparably scaled down stirred tank bioreactor or shake flask. Also, production of gamma-decalactone increased threefold by tripling the oscillatory mixing speed.
The prototype OFR system has been in operation for six months in Dr. Teixeira’s laboratory and is just starting to be optimized.
“Scale up is straightforward, and biopharmaceutical companies are very interested in this approach, too,” he says.