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    SPP 2170: Towards control of population heterogeneity induced variations in process performance during L-phenylalanine production with Escherichia coli
    Upscaling of bioprocesses is often accompanied by loss in productivity compared to well-mixed lab-scale processes. One reason is the formation of population heterogeneity whose mechanistic understanding is still comparably low. During the first funding period (project ProPHet) quantitative understanding of population heterogeneity originating from cell-bioreactor interaction in fed-batch processes with E. coli FUS4 (pF81kan) converting glycerol to L-phenylalanine (L-Phe) was raised. Experimental investigation was done with a quadruple reporter strain monitoring growth, general stress response, oxygen availability and product formation of single cells. Cultivations in a well-mixed stirred-tank bioreactor (STR) were compared with large-scale bioprocess conditions simulated in a scale-down reactor system consisting of a STR and a novel by-pass reactor that is designed as a coiled flow inverter (CFI). Theoretical investigation was done with population balance equations and agent based modeling after generating a non-segregated coarse-grained model. Building upon results from ProPHet, ProPHet2Con is aiming at controlling population heterogeneity induced variations in process performance in the fed-batch processes for L-Phe production. A population heterogeneity level that is beneficial for process performance will be maintained by promoting the abundance of subpopulations with advantageous characteristics. A special focus will be put on the process in the STR-CFI. Prio to implementation of process control, the L-Phe production process will be optimized applying the coarse-grained model and stoichiometric modeling to improve final product titer and productivity, especially in the STR-CFI setup. As a prerequisite for influencing the level of population heterogeneity by automated process control, manual at-line flow cytometry measurement will be replaced by automated real-time flow cytometry (ART-FCM). The ART-FCM setup will be advanced by the previously published process concept segregostat, that allows to control the degree of diversification of a bioreactor population at a predefined setpoint by single-cell physiology-based feeding of the carbon source. Furthermore, model-based soft sensors will implemented to estimate quantities cannot be measured directly. Then, based on the outcome of experimental investigations, strategies will be formulated to influence the level of population heterogeneity by model-based process feedback control following ART-FCM evaluation of single cell physiology. To our knowledge such a coupling between ART-FCM and an advanced model-based process control has so far never been realized. The formulated strategies will be experimentally validated in the fed-batch process for L-Phe production in the STR and the STR-CFI.Once process control for L-Phe production is realized, scale-up potential of the process to pilot-scale will be investigated.
      63
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      213
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    Technologien für Zell- und Gewebekulturen
    Kooperationen: Bioengineering AG, Wald, SchweizBioglobe GmbH, Hamburg Dr. Uwe Marx, TissUse GmbH, Berlin DTL Biotechnology Ltd, Ireland Fraunhofer Einrichtung Marine Biotechnologie, Lübeck Infors AG, Schweiz Ingenieurbüro Dr.-Ing. Schoop GmbH medorex eK, Bovenden Prof. Dr.-Ing. Björn Frahm, FH Lemgo Prof. Dr.-Ing. Dieter Eibl, ZHAW Wädenswil, Schweiz Prof. Dr.-Ing. Hoc Kiem Trieu, Technische Universität Hamburg-Harburg, Institut für Mikrosystemtechnik Prof. Dr.-Ing. Peter Czermak, TH Mittelhessen, Gießen Prof. Dr.-Ing. Thomas Becker, TU München Prof. Dr.-Ing. Volker C. Hass, HS Furtwangen Prof. Dr. Gerardo Catapano, Uni. of Calabria, Italy Prof. Dr. Michael Amling, UKE Hamburg Prof. Dr. Roland Lauster, TU Berlin Prof. Dr. Udo Schumacher, UKE Hamburg ZellWerk GmbH, Eichstädt Zellkulturprozesse zur Wirkstoffproduktion Steuerung von fed-batch-Prozessen Design, Betrieb und Scale-up von Festbettreaktoren mit immobilisierten Zellen Tissue Engineering Herstellung implantierbarer Knorpel-Träger-Konstrukte als Gelenkflächenersatz 3D-Gewebekulturen für Toxizitätstests
      211
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      84
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    SPP 2240: Chemoenzymatic reaction cascade in an All-in-One electrochemical system with in situ supply of H2O2 for biosynthesis in aqueous and organic media
    Chemoenzymatic reaction cascades have a great potential for biosynthesis and sustainability, especially when coupled with the latest advances in electrochemical systems and material sciences. This envisaged project connects the expertise of three truly interdisciplinary groups in biocatalysis, bioprocess engineering, bioelectrochemical systems (BES) and nano-materials in a unique way to address fundamental challenges in developing a novel and individually optimizable platform for hydrogen peroxide-dependent enzymatic cascade reactions. H2O2 plays an important role as a green and powerful co-substrate or co-factor in the biosynthesis of a vast variety of value-added products and can be electrochemically efficiently generated from H2O and O2 as demonstrated in conventional BES with three-electrodes. One of the first objectives of this project is to develop a fully controllable BES for in situ and on-demand synthesis of H2O2 based on a novel All-in-One (AiO) electrode developed recently by one of the project partners. To realize a controlled H2O2 generation in the AiO electrode with high energy efficiency, highly porous carbon foams coated with carbon nanotubes (CNTs) will be designed and tailored by a sophisticated novel CVD process, yielding low density (20 mg/cm3) and high conductive hierarchical porous structure. H2O2 generated in such a controlled way is instantly used by enzymatic oxyfunctionalization in multistep chemoenzymatic reaction cascades. The reaction systems aimed at will include a peroxidase catalyzed sequential hydroxylation towards the synthesis of hydroquinone in aqueous media as well as the simultaneous chemoenzymatic esterification and epoxidation towards epoxidized fatty acid esters in a solvent free organic environment (adapted Prilezhaev reaction). The mentioned reactions will be investigated using both free and immobilized enzymes on functionalized CNTs. This integrated approach is unique and promising in tackling one of the major issues in H2O2 -dependent enzymatic oxyfunctionalization, namely the lowered half-life time of enzyme activity in the presence of H2O2. By integrating the AiO-electrode in an organic reaction medium in situ H2O2 addition will be realized without the addition of water, omitting side reactions (e.g. in the Prilezhaev reaction). With a fine-tunable system in hand, the rates of electrochemical H2O2 synthesis and enzymatic H2O2 consumption can be tightly coupled, enabling a constantly low concentration of H2O2 during the whole cascade reaction. This will open up new possibilities to develop chemoenzymatic reaction cascades with improved enzyme stability and activity and a more stable entire process.
      316
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    SPP 2240: Systems metabolic engineering of Hydrogenophaga pseudoflava for aerobic biosynthesis of fatty acids using CO2 and electron carriers in a novel bioelectrochemical system
    Electrobiotechnology is a promising technology at the interface of electrochemistry and biotechnology to use CO2 and electricity for the microbial biosynthesis of chemicals and fuels in bioelectrochemical systems (BES). An appealing approach in this regard is the conversion of CO2 to CO and the formation of syngas (H2, CO, CO2) electrochemically; the latter is then biologically converted into chemicals or liquid fuels. Although the decoupled bioconversion of syngas has made impressive progresses recently, this technology still has several inherent obstacles such as limited mass transfer of gases into culture medium, low uptake or inefficient transfer of electrons or electron carriers to the microbial host, and safety issues regarding toxicity and explosiveness of the substrates. Furthermore, acetogens as the mostly used microbial hosts for syngas bioconversion have a limited spectrum of products since the production of more complex molecules is outside the metabolic capacity. In this project, the great potential of Hydrogenophaga pseudoflava in the aerobic utilization of syngas and the capacity of engineered H. pseudoflava for the production of fatty acids will be explored in a novel direct electromicrobial production system with in situ and on demand production of H2 and O2 (from water) and CO (from CO2). To develop a systemic and quantitative understanding of the electron transfer and its impact on redox and energy metabolism of this carboxydotrophic bacterium, we will apply metabolomics, flux analysis and quantify kinetic parameters of the wild type and engineered mutants defective in electron transfer, energy and redox metabolism under given gas compositions provided by an optimized BES. The BES will address current limitations of bio-electrochemical systems and gas fermentations as already mentioned above. The gained knowledge will be utilized to set up a first metabolic and electron transfer model of the autotrophic metabolism of H. pseudoflava, especially regarding uptake of the different electron carriers, energy and redox balances. Furthermore, we will engineer H. pseudoflava for the production of fatty acids which represent an ATP and NADPH intensive product class and therefore its overproduction will challenge the metabolism of H. pseudoflava especially under autotrophic conditions. A quantitative analysis of the developed strains will be used to evaluate and refine the metabolic and electron transfer model. This project will open up new possibilities to engineer efficient electromicrobial production strains and to develop improved electro-fermentation.
      158
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      149
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    Subproject of SPP 1934: EnzymAgglo - Multiscale model-based investigation of functional enzyme and protein agglomerates for biotechnological applications. Part 2: From structure to function
    The model based understanding of the structure defining processes of multi-enzymatic systems and protein agglomeration, as well as their relation to function (e.g., activity) has received increasing interest in recent years. With new multi-scaled modeling approaches and increased computational capabilities in recent years (especially GPUs), the detailed investigation and prediction of such macromolecular phenomena comes into reach. To achieve this, we developed a generally applicable multi-scale model framework, termed “Molecular discrete element method (MDEM)”, during the first project phase.Specifically, it focuses on the structural development of such systems depending on process conditions. It applies on the macro-molecular scale (ms and µm) and will be finalized in the remaining time of the first period of the project. The MDEM framework allows investigations of biologically and technically interesting structural phenomena due to its speedup of 5 to 7 orders of magnitude with respect to simulation time and/or scale in comparison to traditional MD. At the same time, it retains the necessary detail on the meso-scale through anisotropic consideration of properties. While, during the first project phase, the framework was established using the multi-enzymatic pyruvate dehydrogenase complex (PDC) as a model system, it has received very positive feedback from other SPP project partners for applications to numerous other processes and phenomena (e.g., protein absorption at the oil-water interface). Initial collaborations have been initiated and are planned to be intensified. The focus of the second project phase will lie on expansion of the MDEM approach with respect to the determination of function from structure; increased size and time scales; and application of developed framework to new processes in collaboration with SPP project partners. To achieve these goals, the MDEM framework will be coupled to population balance modeling (e.g., for experimental validation and parameter studies). Further, protein-protein and protein-interface interaction will be refined and extended to cover density effects (molecular crowding; diffusion limitation), and competetive interaction of multiple partners. Deduction from structure to function will be done by coupling MDEM to computational fluid dynamics / finite volume computation method, including spatially resolved enzymatic reaction and metabolite diffusion kinetics. A further abstraction will take place to enable higher-scale model (termed “MDEM-2”) and push towards the process scale: Stably connected units (e.g., the catalytic core of PDC) are abstracted as one particle and parameterized using the data from MDEM. Experimental feedback for catalytically active multi-enzymatic systems will be obtained in-house, while extensive collaborations with SPP project partners are developing and ongoing, allowing to refine and verify the MDEM(-2) approach on wide variety of process parameters.
      639
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    SPP 2170: Subproject "CaproMix – Development of new and defined co-cultures for bioproduction of caproic acid from carbon dioxide"
    This project brings together well complementary expertise of two academic groups from microbiology and molecular biology (Dr. FR Bengelsdorf) and bioprocess engineering/systems biology (Prof. AP Zeng). The objective is to gain quantitative and fundamental knowledge of synthetic co-culture(s) producing caproic acid (CA) via lactic acid (LA) from CO2 and H2. CA is an appealing C6-compound with a wide range of important applications. In the anaerobic co-culture to be studied an Acetobacterium woodii mutant should produce LA autotrophically and a selected strain from Clostridium kluyveri, Megasphera cerevisiae, and mutants of a newly isolated Caprobacter fermentas should convert LA into CA. The compatibility of different combinations (co-cultures) of these microbes will be first examined in terms of growth physiology (optimal pH, T, nutrient requirement and growth inhibition or enhancement). Metabolic interactions of the selected co-culture will then be studied more quantitatively and in more detail, e.g. regarding kinetics of cell growth, substrate consumption and product formation. For the quantification of cell population a metabolic engineering approach will be applied to generate A. woodii mutants expressing a special fluorogenic protein tag that causes a distinct fluorescence pattern to distinguish cells of different species, along with use of fluorescence-based flow cytometry to quantify the other microbe in the co-culture. A special two-chamber bioreactor system will be used to better study metabolic exchanges and interactions of the two microbes, among others for metabolic flux analysis. Similarly as with the characterization of the co-culture integrated biomolecular and bioprocess engineering approaches will be collaboratively applied to improve the performance of the co-culture for bioprocess development. In addition to extensive genetic and metabolic engineering work planned to improve LA production in A. woodii an adaptive laboratory evolution approach will be implemented to generate a hyper LA producer by suppressing cells that do not produce LA via the use of a LA induced selection pressure. A novel cultivation strategy using a bioelectrochemical system will be applied that allows tuning the in situ electrolytic H2 generation and consequently the metabolism of A. woodii according to the requirement of the LA-consuming and CA-producing strain in the co-culture. Accumulation of toxic LA concentration can be prevented via online measurement of LA which serves as a feedback control parameter for the in situ electrolysis. In summary, this project deals with a number of key scientific questions for developing efficient co-culture technology for the production of value-added products such as lactic and caproic acids from CO2 and regenerative electricity. It bears several methodological and technological innovations which not only help to realize the special goal of this project, but could be also applied to related products.
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