Metabolic Responses to Bioreactor Inhomogeneities: Understanding the Flux to Modified Branched Chain Amino Acids
Final Report Abstract
"Escherichia coli" is an important strain for recombinant protein production in large-scale fed-batch bioprocesses. Due to limited mixing capacities, gradients exist concerning the nutrient and oxygen availability when a certain scale and cell density is reached. In order to investigate the impact of these heterogeneous conditions on the physiologic state of the cells, experiments in a scale-down two-compartment and three-compartment reactor were performed. This concept consists of a stirred tank reactor connected to either one or two plug-flow reactors, which represent conditions at the feeding zone by a combination of oxygen limitation and a high concentration of substrate, and a zone far away from the feeding zone, in which strong starvation conditions occur. It was shown that an oscillatory substrate supply favours the accumulation of the modified branched chain amino acid (mBCAA) norvaline. Initial studies addressed the question, whether an adoption of cells to substrate limiting conditions in an overnight phase is leading to different experimental results in comparison to the standard procedure - an initial batch phase prior to the feeding phase. The relevance of the (common) latter approach seems to be unclear. It can be assumed that in industrial scale fed-batch processes, cells are adopted to substrate limitation during the fed-batch phase before gradients occur at elevated cell densities. An initial exposition of the culture to glucose-limited fed-batch conditions before the exposure to oscillatory conditions resulted in the accumulation of metabolites, like lactic, formic, succinic, and malic acid, while no accumulation of these acids was detected in cultivations where the feeding was started after a batch phase. An increased accumulation at oscillating substrate availability as it was reported before was not observed in the two-CR, but in the three-CR for norvaline, norleucine and isoleucine. When a recombinant leucine-rich protein was expressed, the incorporation of norleucine (and other mBCAAs) was increased. While the methionine content was reduced, the portion of incorporated norleucine was more than doubled (0.57 %). In order to improve the understanding of the changed carbon distribution around the branch-point intermediate pyruvic acid in "E. coli", when cells were exposed to oscillating conditions, the rapid sampling unit BioScope was applied. At the entrance of it, 13C-labeled glucose was added to a sample from either a homogeneous single-CR or heterogeneous two-CR cultivation. In both cases, oxygen limitation was achieved. The results showed a much lower degree of incorporation for several intermediates (fumaric and malic acid) of the tricarbonic acid cycle (TCA). That is a reduced flux throughout the TCA cycle was seen when the culture was exposed to oscillatory conditions. In this case, the incorporation into the amino acids aspartate and threonine was also much lower, which supports the hypothesis of a shortcut from pyruvic acid directly towards the mBCAA pathway. As consequence, the incorporation of 13C-labeled carbon into aspartate and threonine is reduced and enhanced for isoleucine and the mBCAAs norvaline, norleucine and beta-methylnorleucine. This incorporation was tremendously higher when oxygen limitation occurred in parallel to glucose excess rather than only glucose excess conditions. Hence, it can be assumed that adaptation to oxygen limitation might play a pivotal role in the redirection of fluxes directly from pyruvic acid towards the BCAA synthesis as previously suggested. A major change of protein expression was seen for the isocytrate lyase ("aceA"), which is involved in acetate re-assimilation and the glyoxylate shunt. All in all, the expression experiments do not indicate a higher expression of enzymes of reactions towards typical products of oxygen limitation (like formate, methanol), but a reduced expression of several enzymes using TCA intermediates as substrates. Although the response to oscillatory conditions of "E. coli" are rather weak in terms of the accumulation of (m)BCAAs, the response to glucose excess is changed when the cell is exposed to oscillatory conditions. Higher fluxes towards the mBCAA under conditions of protein expression are leading to a higher incorporation of non-canonical amino acids. However, the experimental design of oscillatory conditions has a tremendous impact on the results. In order to mimic industrial cases, an exact reproduction of cultivation conditions is a prerequisite for achieving relevant results.
Publications
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The branched chain amino acid synthesis in Escherichia coli under large scale conditions in a two‐compartment reactor. European Conference of Applied Microbiology (ECAB), September 2011, Berlin
E. Brand, Baudis M, Junne S, Neubauer P
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Consistent bioprocess development in scaled‐down fed‐batch cultivation systems: combining suitable hardware and software approaches. Annual Meeting of the Society of Industrial Microbiology and Biotechnology (SIMB) 13.‐16.08.2012, Washington, DC
S. Junne, Brand E, Cruz Bournazou NM, Knepper A, Neubauer P
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Consistent fed‐batch bioprocess development in the µL to L‐scale. The BioProcess Summit, 21.‐23.08.2012, Boston, MA
S. Junne, Brand E, Cruz Bournazou NM, Knepper A, Neubauer P
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„Consistency of scale‐up from bioprocess development to production”,2012. In “Biopharmaceutical Production Technology” Vol. 2. G Subramanian (ed.). Wiley VCH Weinheim. Pp. 513‐543
S. Junne, Klingner A., Itzeck D., Brand E., Neubauer P.
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Integration of metabolic flux analysis into a scale‐down two‐compartment bioreactor study of Escherichia coli cultivations. 9th European Congress of Chemical Engineering (ECAB), April 21–24, 2013, Hague, The Netherlands
E. Brand, Junne S, Lemoine A, Trippel S, Spann R, Neubauer P.
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Recombinant Protein Production: A Comparative view on host physiology (Laupheim, Germany). New Biotechnology 2013; 30 (5), 405‐409
C. Reitz, Li J, Neubauer P.
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2014. Use of Sensors in a Scale‐Down Simulator. Studying the Impact of Industrial‐Scale Inhomogeneities on Microbial Cultures. Gen Engin News 34 (6), 32‐33 (March 15, 2014)
S. Junne, Klingner A, Itzeck D, Neubauer P.
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Simulating large scale conditions in a scale‐down bioreactor: impacts on the cell physiology of Escherichia coli. 3rd Int. BioProScale Symposium, April 2‐4, 2014, Berlin
C. Reitz, Brand E, Lu P, Spann R, Trippel S, Junne S, Neubauer P
