Mikroaerophile Alginatproduktion mit Azotobacter vinelandii

In this study, growth characteristics of the nitrogen fixing bacterium Azotobacter vinelandii as well as some factors controlling the alginate production by this strain were quantitatively investigated. From a quantitative examination of growth parameters, respiration and morphological changes, relationships between the different essential processes, i.e. nitrogen fixation, respiration intensity, PHB biosynthesis and alginate formation, were established. The first part of this study was focused on the production of alginate under controlled-microaerophilic conditions to examine the suitable oxygen concentration profile in respect to quantitative alginate formation for this strain. Testing a pO2 range of 0-10 air saturation, showed that intermediate pO2 values (2.5-5) were optimal for alginate and biomass production. Above and below these values, the bacterium either wasted the carbon and energy source in respiration (CO2) or in PHB biosynthesis (44-58 of the biomass). Thus, efficient conversion of carbon source to alginate is achieved only if the DOT is accurately controlled at an intermediate value. To lower the extreme O2 sensitivity, different nitrogenous rich compounds were added to the mineral medium. Surprisingly, the cells exhibited a pronounced oxygen sensitivity even in the presence of complex nitrogenous materials. Furthermore, higher alginate yield (Yalg/X) was obtained in nitrogen free medium, and for the purpose of medium simplicity the addition of nitrogenous compounds was not considered. To gain more insights toward a better understanding of the inhibitory effect of oxygen towards nitrogenase and its relation to alginate biosynthesis, respiratory protection - one of the two mechanisms for nitrogenase protection - was impaired through phosphate limitation. This was performed in a pO2 controlled bioreactor (2.5-3) as well as in shaked flasks.Im Mittelpunkt dieser Arbeit steht die Untersuchung der Wachstumseigenschaften des stickstoffixierenden Bakterienstammes Azotobacter vinelandii sowie die Quantifizierung einiger die Alginatbiosynthese beeinflussender Faktoren. Quantitative Untersuchungen vor Wachstumsparameter, Atmung und morphologischen Veränderungen ermöglichen die Klärung des Zusammenhanges zwischen essentiellen Prozessen wie Stickstoffixierung, Atmungsaktivität, PHB-Biosynthese und Alginatproduktion. Das für die quantitative Alginatproduktion optimale Sauerstoffkonzentrationsprofil wurde unter kontrollierten mikroaerophilen Bedingungen ermittelt. Untersuchungen zum Einfluß verschiedener Konzentration von gelöstem Sauerstoff (pO2: 1-10) sowohl im Hinblick auf die Alginatausbeute als auch in Bezug auf die Biomasse ergaben einen Sauerstoffbereich von 2.5-5 Ein geringer oder höherer pO2 führte zum Verlust von Kohlenstoff zugunsten der PHB-Synthese (44-58 der Biomasse) bzw. zur Veratmung zu CO2. Die effiziente Umwandlung der Kohlenstoffquelle zu Alginat ist nur durch die genaue Kontrolle einer intermediären Gelöst-Sauerstoffkonzentration möglich. Um die extreme Empfindlichkeit des Stammes gegenüber Sauerstoff zu senken, wurden dem Mineralsalz-Medium verschiedene stickstoffreiche Verbindungen zugegeben. Es konnte überraschenderweise gezeigt werden, daß die Zellen sogar in Gegenwart von Stickstoffverbindungen eine deutliche Sauerstoffempfindlichkeit aufwiesen. Außerdem wurde im stickstofffreien Medium eine höhere Alginatausbeute (Yalg/x) erzielt, so daß zur Vereinfachung des Mediums auf die Zugabe von Stickstoffverbindungen verzichtet wurde

New technologies for enzyme engineering: Combining computational predictions and automated experimental feedback

The targeted design and optimization of novel enzymes and enzymatic reaction cascades increasingly demands a close connection between rational design, computational prediction and experimental feedback. In recent years, lots of effort have been put on increasing the throughput of experimental results, however, this approach frequently tends to stick in local minima and unsatisfying performance improvement despite considerable screening efforts. Contrary, model-based computational predictions, despite increasing available computation power, need to introduce severe simplifications and therefore will continue to lack accuracy and perfect predictability in the foreseeable future. The interplay of thorough model-based understanding, automated experimental feedback and, based on the latter, refinement of model predictions using for example machine learning methods, will in the near future become an important approach to combine the best of the two worlds. Ultimately, this provides potential to boost highly efficient automated or semi-automated design of new enzymatic properties in the scope of a “fourth wave” of enzyme engineering. We present a new integrated directed evolution framework to achieve this simulation-experimental feedback loop, called “Feedback Guided Enzyme Optimization” (FEO). The implementation includes the setup of a suitable simulation back-end, robot-based experimental generation of mutants and evaluation of their performance [1], and finally feedback to the simulation in order to close the loop and verify and refine the quality of the predictions.Focus is laid on thorough statistical analysis of both prediction and experimental results, in order to tune false positive vs. false negative error rate, depending on experimental conditions: This includes, e.g., availability of time, ingredients, parallel workflows and distortions (random noise and potential systematic deviations) in both experimental and simulation setups. The framework is being implemented in an automated robotic setup. We demonstrate results on three exemplary enzymatic systems: Firstly, GFP is employed as a simple role model to demonstrate the looping principle. The second example, aspartokinase III (AK3), is a key enzyme for the biosynthetic production of amino acids and derivatives thereof. Its activity is naturally limited by its own downstream products, e.g., lysine. Simulated predictions of the sensitivity of AK3 towards lysine have been compared to experimental data. This allowed a significant (p\u3c0.05) simulation-based discrimination of highly resistant versus non-resistant variants. Determination of new lysine resistant mutants by multiple point mutations is performed within few dozen of iterations. The obtained candidates were validated, showing that new Lys-resistant variants can be obtained using the new workflow without special a priori knowledge or extensive (random) screening. The third and most sophisticated enzyme system is the pyruvate dehydrogenase complex (PDC) which involves interesting features like shielding of reaction intermediates, renewal of co-factors, self-assembly, modularity and others. Based on recently published models of PDC by our group [2-3] and in collaborations [4], we demonstrate how the dynamic self-assembly of mutants of PDC and structurally similar enzymes complexes can be predicted, iteratively refined and in the future used for the creation of new enzyme cascades. This presented framework is expected to have large impact on design and evolution of novel biomolecules and biosystems. [1] Wurm, M., Ilhan, S., Jandt, U., Zeng A.-P. (2019). Direct and Highly Sensitive Measurement of Fluorescent Molecules in Bulk Solutions using Flow Cytometry. Analyt. Biochem. 570, 32-42. [2] Hezaveh, S., Zeng, A. P., Jandt, U. (2018). Enzyme Interaction in Human Pyruvate Dehydrogenase Complex: Full Complex Simulation. Journal of Chemical Information and Modeling, 58(2), 362-369. [3] Hezaveh, S., Zeng, A. P., Jandt, U. (2017). Investigation of Core Structure and Stability of Human Pyruvate Dehydrogenase Complex: A Coarse-Grained Approach. ACS Omega, 2(3), 1134-1145. [4] Depta, P.N., Jandt, U., Dosta, M., Zeng, A.-P., Heinrich, S. (2019). Toward Multiscale Modeling of Proteins and Bioagglomerates: An Orientation-Sensitive Diffusion Model for the Integration of Molecular Dynamics and the Discrete Element Method. J. Chem. Inf. Model. 59(1), 386-398

Co-cultivation of Lactobacillus zeae and Veillonella criceti for the production of propionic acid

In this work a defined co-culture of the lactic acid bacterium Lactobacillus zeae and the propionate producer Veillonella criceti has been studied in continuous stirred tank reactor (CSTR) and in a dialysis membrane reactor. It is the first time that this reactor type is used for a defined co-culture fermentation. This reactor allows high mixing rates and working with high cell densities, making it ideal for co-culture investigations. In CSTR experiments the co-culture showed over a broad concentration range an almost linear correlation in consumption and production rates to the supply with complex nutrients. In CSTR and dialysis cultures a strong growth stimulation of L. zeae by V. criceti was shown. In dialysis cultures very high propionate production rates (0.61 g L-1h-1) with final titers up to 28 g L-1 have been realized. This reactor allows an individual, intracellular investigation of the co-culture partners by omic-technologies to provide a better understanding of microbial communities. © 2013 Bel-Rhlid et al

Simultaneous production of 1,3-propanediol and n-butanol by clostridium pasteurianum: in situ gas stripping and cellular metabolism

Clostridium pasteurianum is a promising producer of the bulk chemicals 1,3-propanediol and n-butanol (BuOH), with significantly different product patterns and physiology from other typical Clostridia. Nevertheless, its growth and product formation are ultimately limited by the accumulation of inhibiting products, especially by BuOH. In this study, we implemented in situ gas stripping for BuOH removal and compared the stripping performance of external nitrogen (N2) and fermentation effluent gas (FG) from the process itself. Gas stripping was studied in fermentations of glycerol and a mixture of glycerol and glucose. In general, N2 exhibits favourable physical properties to strip out BuOH from an aqueous phase. However, in situ removal of butanol in C. pasteurianum culture with N2 stripping strongly perturbs the culture conditions such as the redox potential and thus the physiology of the microorganism, leading to enhanced formation of organic acids, especially in cosubstrate fermentation, whereas the use of FG does not show such perturbations. In an effort to explore the use of FG for gas stripping the effects of FG circulation rate and stirring speed of the bioreactor on BuOH stripping efficiency and the fermentation performance were studied in more detail. Mass transfer coefficient (kSa) of BuOH in the bioreactor was also characterized at different gas circulation rates and stirring speeds. In a fermentation of glycerol with FG stripping at a relatively high gas flow rate (7 vvm) as high as 39.2 g/L BuOH (total) and 53.7 g/L 1,3-PDO can be simultaneously produced. The results are discussed in view of further process optimization and scale up

Microbial cell factories for diol production

Diols are compounds with two hydroxyl groups and have a wide range of appealing applications as chemicals and fuels. In particular, five low molecular diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO), 1,3-butanediol (1,3-BDO), and 1,4-butanediol (1,4-BDO), can be biotechnologically produced by direct microbial bioconversion of renewable materials. In this review, we summarize recent developments in the microbial production of diols, especially regarding the engineering of typical microbial strains as cell factory and the development of corresponding bioconversion processes

Co-cultivation of Lactobacillus zeae and Veillonella criceti for the production of propionic acid

In this work a defined co-culture of the lactic acid bacterium Lactobacillus zeae and the propionate producer Veillonella criceti has been studied in continuous stirred tank reactor (CSTR) and in a dialysis membrane reactor. It is the first time that this reactor type is used for a defined co-culture fermentation. This reactor allows high mixing rates and working with high cell densities, making it ideal for co-culture investigations. In CSTR experiments the co-culture showed over a broad concentration range an almost linear correlation in consumption and production rates to the supply with complex nutrients. In CSTR and dialysis cultures a strong growth stimulation of L. zeae by V. criceti was shown. In dialysis cultures very high propionate production rates (0.61 g L-1h-1) with final titers up to 28 g L-1 have been realized. This reactor allows an individual, intracellular investigation of the co-culture partners by omic-technologies to provide a better understanding of microbial communities. © 2013 Bel-Rhlid et al

Metabolic network analysis and experimental study of lipid production in Rhodosporidium toruloides grown on single and mixed substrates

Background: Microbial lipids (triacylglycerols, TAG) have received large attention for a sustainable production of oleochemicals and biofuels. Rhodosporidium toruloides can accumulate lipids up to 70% of its cell mass under certain conditions. However, our understanding of lipid production in this yeast is still much limited, especially for growth with mixed substrates at the level of metabolic network. In this work, the potentials of several important carbon sources for TAG production in R.toruloides are first comparatively studied in silico by means of elementary mode analysis followed by experimental validation. Results: A simplified metabolic network of R.toruloides was reconstructed based on a combination of genome and proteome annotations. Optimal metabolic space was studied using elementary mode analysis for growth on glycerol, glucose, xylose and arabinose or in mixtures. The in silico model predictions of growth and lipid production are in agreement with experimental results. Both the in silico and experimental studies revealed that glycerol is an attractive substrate for lipid synthesis in R. toruloides either alone or in blend with sugars. A lipid yield as high as 0.53 (C-mol TAG/C-mol) has been experimentally obtained for growth on glycerol, compared to a theoretical maximum of 0.63 (C-mol TAG/C-mol). The lipid yield on glucose is much lower (0.29 (experimental) vs. 0.58 (predicted) C-mol TAG/C-mol). The blend of glucose with glycerol decreased the lipid yield on substrate but can significantly increase the overall volumetric productivity. Experimental studies revealed catabolite repression of glycerol by the presence of glucose for the first time. Significant influence of oxygen concentration on the yield and composition of lipids were observed which have not been quantitatively studied before. Conclusions: This study provides for the first time a simplified metabolic model of R.toruloides and its detailed in silico analysis for growth on different carbon sources for their potential of TAG synthesis. Experimental studies revealed the phenomenon of catabolite repression of glycerol by glucose and the importance of oxygen supply on the yield and composition of lipids. More systematic studies are needed to understand the mechanisms which should help to further optimize the lipid production in this strain of industrial interest