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

Automated, simulation-assisted and feedback-guided biomolecular engineering

The modification and even more de novo construction of novel enzymatic and multienzymatic bioreaction cascades is of high interest for biotechnological and medical applications [1]. Two main strategies have been established and evolved significantly in the recent years to engineer and optimize such enzymatic systems. First, rational approaches based on structural model descriptions; second, high-throughput screening of numerous, often randomly generated variants. Ideally, both methods should be combined and complement each other in an automated manner, involving minimal manual effort. Key to such successful interconnection is the combination of a reliable model based understanding of the enzymatic systems; prediction of the relevant enzymatic properties based on it; and feedback from experimental data to refine the model-based predictions. The latter refinement should ideally be implemented automatically using machine learning. We introduce a new integrated concept and its automatized integration to achieve this simulationexperimental feedback loop and especially to overcome the problem of “combinatorical explosion” when targeting multiple modification sites in parallel. We termed the approach feedback-guided enzyme optimization (FEO) and apply it to two exemplary enzymatic systems of interest. The first enzyme, aspartokinase III (AK3) of E. Coli, is a bottleneck enzyme in the lysine biosynthetic pathway. It is naturally inhibited via an allosteric conformation transition caused by its own downstream product, lysine (amongst other inhibitors). During many decades, this enzyme has been heavily engineered to overcome this inhibition; hence many data is available for this enzyme system. Simulation predictability of AK3 sensitivity to lysine has been compared to experimental own and literature data, allowing for a significant (p\u3c0.05) simulationbased discrimination of highly resistant versus non-resistant variants. Determination of new lysine resistant mutants by multiple point mutations is performed within few dozen (usually \u3c100) iterations, which is computationally feasible using the presented multi-scaled approach. The obtained candidates are statistically evaluated and experimentally validated, showing that new Lys-resistant variants can be obtained using the new workflow without special a priori knowledge or extensive (random) screening. The second exemplary enzyme system of interest is the pyruvate dehydrogenase complex (PDC), a highly ordered and so far not well understood enzyme complex consisting of more than 100 enzymes and integrating all properties that are typically necessary for efficient enzymatic reaction cascades, like shielding of reaction intermediates, renewal of co-factors and arrangement of efficient enzymatic clusters. Its very special modular construction, mediated by linker arms, enables flexible exchange and modification of functional parts while maintaining self-assembly capability, and regulation. Based on a recently published novel model of the catalytic core of PDC [2-3], we demonstrate how the dynamic self-assembly of mutants of PDC and structurally similar enzymes complexes can be predicted, iteratively refined and used for the creation of new highly active enzyme cascades. The overall approach is currently being fully integrated in an automated robotic setup. It is expected to open up new possibilities for a more global optimization of enzymes and enzyme cascades. [1] Jandt U., You C., Zhang Y. H.-P., Zeng A.P. (2013), In: Adv. Biochem. Eng. Biotechnol. 137, 41-65. [2] Hezaveh S., Zeng A.P., Jandt U. (2016). J. Phys. Chem. B, 120(19), 4399-4409. [3] Hezaveh S., Zeng A.P., Jandt U. (2016). Investigation of Core Assembly and Stability of Human Pyruvate Dehydrogenase Complex: a Coarse-grained Approach , ACS Omega, in print, doi 10.1021/acsomega.6b00386. [4] Guo J., Hezaveh S., Tatur J., Jandt U. (2016). “Reengineering of human pyruvate dehydrogenase complex: from disintegration to highly active agglomerates”, Biochem. J., 474(5), 865-875. [5] Castellana, M., Wilson, M.Z., Xu, Y., Joshi, P., Cristea, I.M., Rabinowitz, J.D., ... & Wingreen, N. S. (2014). Nature Biotechnol, 32(10), 101

Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation

The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils. In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.</p

Criteria for bioreactor comparison and operation standardisation during process development for mammalian cell culture

Platas Barradas O, Jandt U, Da Minh Phan L, et al. Criteria for bioreactor comparison and operation standardisation during process development for mammalian cell culture. BMC Proceedings. 2011;5(8)

3rd Helmholtz Open Science Forum „Helmholtz in the German National Research Data Infrastructure (NFDI)“

To promote dialogue on the National Research Data Infrastructure (NFDI) in the Helmholtz Association, the Helmholtz Open Science Office hosted two digital Forums in May and December 2021. The office has organized a third Forum on the topic on June 22, 2023. The objective of this event was to offer insights into the NFDI activities within the Helmholtz Association, presented from the internal perspectives of the Centers. Multiple Helmholtz Centers shared their experiences, fostering an interactive environment for questions and discussions. Furthermore, there were contributions highlighting the Base4NFDI basic service consortium

Characterisation of cultivation of the human cell line AGE1.HN.AAT

Skerhutt EM, Scholz S, Niklas J, et al. Characterisation of cultivation of the human cell line AGE1.HN.AAT. BMC Proceedings. 2011;5(8)

Towards recombinantly produced milk proteins: Physicochemical and emulsifying properties of engineered whey protein beta-lactoglobulin variants

DFG, 273937032, SPP 1934: Dispersitäts-, Struktur- und Phasenänderungen von Proteinen und biologischen Agglomeraten in biotechnologischen ProzessenBMBF, 031B0222, Basistechnologie Nachwuchsgruppe "Multiskalige Modellierung und Modifikation von Multienzymkomplexen als Basistechnologie für zellfreie Reaktionskaskaden" (II

ReSurveyGermany: Vegetation-plot time-series over the past hundred years in Germany

Vegetation-plot resurvey data are a main source of information on terrestrial biodiversity change, with records reaching back more than one century. Although more and more data from re-sampled plots have been published, there is not yet a comprehensive open-access dataset available for analysis. Here, we compiled and harmonised vegetation-plot resurvey data from Germany covering almost 100 years. We show the distribution of the plot data in space, time and across habitat types of the European Nature Information System (EUNIS). In addition, we include metadata on geographic location, plot size and vegetation structure. The data allow temporal biodiversity change to be assessed at the community scale, reaching back further into the past than most comparable data yet available. They also enable tracking changes in the incidence and distribution of individual species across Germany. In summary, the data come at a level of detail that holds promise for broadening our understanding of the mechanisms and drivers behind plant diversity change over the last century

Investigation of Core Structure and Stability of Human Pyruvate Dehydrogenase Complex: A Coarse-Grained Approach

The human pyruvate dehydrogenase complex (hPDC) is a large macromolecular machine, and its unique structural and functional properties make it a versatile target for manipulation aiming for the design of new types of artificial multienzyme cascades. However, model-based and hence systematic understanding of the structure–function relationship of this kind of complexes is yet poor. However, with new structure data, modeling techniques, and increasing computation power available, this shortfall is about to cease. Recently, we have built new atomistic models of E2/E3BP, the two subunits of the massive hPDC core. Here, we present developed coarse-grained models of the same, on the basis of which we built and simulated the full core of hPDC, which is so far the first simulation of the catalytic core of any member in the branched-chain α-keto acid dehydrogenase complex family. We explored the stability of two previously proposed substitutional models of hPDC core: 40E2+20E3BP and 48E2+12E3BP. Our molecular dynamics simulations showed a higher stability and sphericity for the second model. Our core’s radius of gyration was found to be in strong agreement with previously published experimental dynamic light scattering (DLS) data. Finally, in the direction of our experimental effort to design active minimized complexes, we simulated C-terminal truncated E2/E3BP cores of different lengths, which clearly showed the instability of the core assembly and symmetry due to subunit separations. This correlated very well with the findings of our experimental investigations by analysis of DLS data for variable truncation lengths. The use of polarizable water and an increased total ion concentration did not lead to a substantially different initial stability of the truncated mutants compared to that of standard MARTINI water; however, a different rearrangement behavior of the fragments was observed. The obtained structure models will serve as a basis for full complex simulations in the future, providing the possibility for the model-assisted targeted manipulation of such a complex enzymatic system