Bioeconomy comprises the economic utilization of renewable biological resources and their sustainable conversion. Among these resources, plants occupy a key position as they convert water, carbon dioxide and sun light into biomass. However, it remains challenging to access plant- based biomass for the sustainable and economic conversion into bioeconomically relevant products. A possible strategy to accomplish this challenge is the application of naturally occurring plant biomass-degrading enzymes, which also includes the identification of novel enzymes with relevant activities or properties and their subsequent application. Consequently, a prediction pipeline for the identification of such enzymes was established within the BioSC BOOST FUND project BIOExpresSPro. By combining manually curated hidden markov models to query reference databases and detailed visualization of intermediate results, candidate sequences exhibiting potentially desired properties, e. g. thermo- and solvent-tolerance, can now be identified.
Substantial amounts of these enzymes have to be provided to enable economic industrial processes. Thus, the implementation of heterologous expression technology is necessary. This comprises both, the production and secretion of the desired enzymes. Therefore, a novel synthetic biology strategy was employed enabling high-throughput cloning, high-level expression and secretion of target enzymes in different microbial chassis organisms. As proof of concept, expression libraries of the cutinase of Fusarium solani pisi with more than 100 secretion signal sequences were generated and employed to optimize cutinase production in the model organisms Escherichia coli and Corynebacterium glutamicum. Furthermore, gene expression was optimized by improving vector backbones through directed evolution. Additionally, directed evolution was used to optimize a selected secretion signal sequence for improved expression levels. Novel biosensors have also been developed in BIOExpresSPro to allow monitoring the extent of protein secretion via the general secretion (Sec) pathway by C. glutamicum. These Sec biosensors were shown to respond to the secretion of various heterologous target proteins in a dose dependent manner. The suitability of the novel Sec biosensors for the fast and efficient high throughput optimization of the secretory production of heterologous proteins by C. glutamicum has been demonstrated. In parallel, a novel fluorescence protein-based biosensor for the analysis of protein secretion and localization in E. coli was established. This biosensor was shown to be able to determine the secretion rate from cytoplasm to periplasm as well as the localization of the chosen target protein.
On the level of process development, thorough selection of bioprocess conditions was shown to be crucial for protein secretion efficiency, as non-optimal conditions lead to substantial loss regardless of the selected signal peptide. Therefore, an automated Mini Pilot Plant micro-bioreactor system with higher cultivation throughput has been developed, validated and applied successfully for strain screening under well-characterized bioprocess conditions.
The results obtained and the technologies newly developed within BIOExpresSPro undoubtedly contribute to accelerate enzyme identification and enable high-throughput cloning, expression and secretion of industrially important enzymes.
Participating Core Groups
Prof. Dr. Karl-Erich Jaeger, Dr. Thomas Drepper; Institute of Molecular Enzyme Technology (IMET); Heinrich-Heine-University Düsseldorf
Dr. Achim Heck (IMET)
Heinrich Heine University Düsseldorf
Forschungszentrum Jülich GmbH
phone: +49 (0) 24 61 - 61 41 73
Prof. Dr. Michael Bott, Prof. Dr. Roland Freudl; Institute of Bio- and Geosciences: Biotechnology (IBG-1, Systemic Microbiology); Forschungszentrum Jülich GmbH.
Prof. Dr. Wolfgang Wiechert, Prof. Dr. Marco Oldiges; Institute of Bio- and Geosciences: Biotechnology (IBG-1, Systems Biotechnology); Forschungszentrum Jülich GmbH.
Prof. Dr. Björn Usadel; Institute of Biology I (Botany/Molecular Genetics): Plant Walls, Metabolism & Bioinformatics; RWTH Aachen University.
Prof. Dr. Ulrich Schwaneberg, Dr. Anna Joelle Ruff; Institute of Biotechnology (Bio VI, Biotechnology); RWTH Aachen University.
1.1.2014 – 31.12.2016
The total budget of BIOExpresSPro is € 798.586,00. BIOExpresSPro is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Innovation, Science and Research of the German State of North Rhine-Westphalia.
Freier, L, Hemmerich, J, Schöler, K, Wiechert, W, Oldiges, M and von Lieres, E (2016). Framework for kriging-based iterative experimental analysis and design: Optimization of secretory protein production in corynebacterium glutamicum. Engineering in Life Sciences 16(6): 538-549.
Heck, A and Drepper, T (2017). Engineering photosynthetic α-proteobacteria for the production of recombinant proteins and terpenoids. Modern topics in the phototrophic prokaryotes: Environmental and applied aspects. Hallenbeck, P.C. Cham, Springer International Publishing: 395-425.
Hemmerich, J, Rohe, P, Kleine, B, Jurischka, S, Wiechert, W, Freudl, R and Oldiges, M (2016). Use of a sec signal peptide library from bacillus subtilis for the optimization of cutinase secretion in corynebacterium glutamicum. Microbial Cell Factories 15(1): 208.
Loehrer, M, Vogel, A, Huettel, B, Reinhardt, R, Benes, V, Duplessis, S, Usadel, B and Schaffrath, U (2014). On the current status of phakopsora pachyrhizi genome sequencing. Front Plant Sci 5: 377.
MacKellar, D, Lieber, L, Norman, JS, Bolger, A, Tobin, C, Murray, JW, Oksaksin, M, Chang, RL, Ford, TJ, Nguyen, PQ, Woodward, J, Permingeat, HR, Joshi, NS, Silver, PA, Usadel, B, Rutherford, AW, Friesen, ML and Prell, J (2016). Streptomyces thermoautotrophicus does not fix nitrogen. Scientific Reports 6: 20086.
Torra, J, Burgos-Caminal, A, Endres, S, Wingen, M, Drepper, T, Gensch, T, Ruiz-Gonzalez, R and Nonell, S (2015). Singlet oxygen photosensitisation by the fluorescent protein pp2fbfp l30m, a novel derivative of pseudomonas putida flavin-binding pp2fbfp. Photochem Photobiol Sci 14(2): 280-287.