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Production of Terpenoids in Escherichia coli: Optimizing the Metabolic Network and the Production Process

Authors: 
Wagner, S. G., Technical University of Munich

Terpenoids are functionalized terpenes, which are naturally synthetized compounds formed by the ligation of isoprene molecules. They have a wide range of applications in the food, cosmetic, biotechnology and pharmaceutical industry. Lycopene, taxoids and artemisinin are some prominent examples of terpenoid molecules with remarkable applications. The microbiological terpenoid production is a relatively new field of research.  Consequently, much work has yet to be done to improve current experimental product yields and productivities. Here, we report some achievements on the strain and process optimization of the E. coli-based taxoids production.

Firstly, we developed a protocol for Metabolic Engineering which guides the development and improvement of both the production strain and its subsequent usage in a bioreactor [Springer Protocols Handbooks, Chapter: “Computer-guided Metabolic Engineering”, http://dx.doi.org/10.1007/8623_2015_118]. The protocol provides clear advices on the proper order of applying many standard computational methods widely used nowadays for rational Metabolic Engineering. By applying the tools described in the protocol, we could identify different carbon sources maximizing various performance criteria, e.g., carbon yield and profit. Additionally, we could reduce carbon leakage via addition of a synthetic non-oxidative glycolysis, as well as identify optimal process parameters. The in silico process optimization was performed by using experimental batch data at different production temperatures and time point of induction to construct a dynamic flux balance analysis model capable of predicting system behavior at new, untested conditions. By doing this, it was possible to find optimal process parameters that increased the highest experimental productivity by 25%. Further improvements of current host strain require a deep understanding of the metabolic stress induced by the expression of large enzyme cascades, typically required for the synthesis of complex taxoids. For that purpose, we introduced a reporter plasmid expressing the fluorescent protein mCherry under the control of a housekeeping promoter. Additionally, we introduced a second plasmid that provides a controllable load. This system enables us to systematically analyse genes regarding their replicative, transcriptional, and translational burden. A mathematical model which will not only consider metabolic fluxes but also transcriptional and translational processes is being developed and will be validated based on experimental data. By using this model, we will be able to calculate and predict energy and resource requirements for a heterologous burden and consequently, estimate to what extent inserted genes can be expressed.  The developed model should also be useful at proposing genetic modifications as well as process parameters for resource rearrangement and energy delivery to accomplish the burden.