Control systems are ubiquitous in modern day technology. In industrial contexts, these systems ensure that relevant variables remain within a desirable range to keep processes running safely and efficiently. A vast array of control strategies exists, and it is not uncommon to combine different types of controllers to improve performance. For instance, high-level controllers based on mathematical modeling of a given process are routinely combined with low-level controllers, such as the widely used Proportional–Integral–Derivative (PID) controller, which does not rely on models and instead uses real-time feedback to adjust control actions.
Combining different controllers is not, however, a straightforward endeavor. In biosynthesis plants, where engineered bacteria are used to produce desired products, the concentrations of specific substances inside the microorganisms represent important control variables. Not only are these variables difficult to measure accurately but control systems must also address inaccuracies between the mathematical models used and the actual process. Addressing these process-model mismatches (PMMs) is a significant challenge in controlling biosynthetic processes.
Fortunately, a research team led by doctoral student Tomoki Ohkubo from the Graduate School of Science and Technology at Nara Institute of Science and Technology and Associate Professor Katsuyuki Kunida from Fujita Health University, Japan, has been actively investigating a promising solution to this problem. In their latest study published in volume 14 of Scientific Reports on November 18, 2024, they present a convincing proof-of-concept for a hybrid 'in silico/in-cell' controller (HISICC). The team's technology combines a computer-driven, model-based optimization controller with a feedback control mechanism engineered directly into an Escherichia coli (E. coli) bacteria strain engineered for producing fatty acids. Mr. Tomoki Ohkubo from the Nara Institute of Science and Technology also contributed to the research and was the lead author of the study.
To demonstrate the potential of HISICC, the team compared three different control strategies for the E. coli-based synthesis of fatty acids via simulations. The first approach used a previously developed "FA2" strain, which has an engineered regulator that can drive up the production of the key "bottleneck" enzyme acetyl-CoA carboxylase (ACC) when induced by isopropyl-β-D-thiogalactopyranoside (IPTG). While the overexpression of ACC increases fatty acid yield, excessive strain on the bacteria can lead to cytotoxicity. As a result, this "no brakes" approach is the most rudimentary form of control technique.
In contrast, the HISICC approach uses a previously developed "FA3" strain, which contains an in-cell feedback loop. "In this design, if the ACC level becomes too high, increased levels of a responsive transcription factor trigger the production of a lactose repressor protein, which in turn represses ACC overexpression," explains Mr. Ohkubo, "Meanwhile, like in FA2, IPTG can be administered externally to drive up ACC expression." The third and final approach employed a newly designed strain called "FA4." This bacterial strain did not have a feedback loop but instead contained a second "input channel" to drive up the production of lactose repressor protein. Simply put, FA4 has an externally controlled "brake system," unlike FA2.
The team carefully developed mathematical models for each bacterial strain and conducted a series of simulations addressing different types of PMMs. As expected, the FA3 and FA4 strains performed better than FA2. This improved performance was attributed to the ability to tune down ACC overexpression before bacterial strains are negatively affected, meaning that enforcing a higher initial ACC expression was possible, leading to a higher initial yield. Notably, among the three methods tested, the HISICC approach (FA3) achieved the highest fatty acid yield. "Our findings demonstrate the potential of HISICC with intracellular biosensing as a solution to the PMM problem," remarks Dr. Kunida. "Considering that the in-cell controller used in this study is rather simple in structure and crude in operation, we expect that a more advanced in-cell controller capable of differential or second-order differential control would reduce response times and markedly improve performance."
Together, the techniques and results featured in this study could form the basis for new control strategies in complex industrial bioprocesses. By improving the production efficiency of fuels and important chemicals, the HISICC approach has the potential to reduce costs and minimize the environmental impact associated with these processes.
