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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.
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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.
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