Dynamic Metabolic Control: From the Perspective of Regulation Logic

Abstract

Establishing microbial cell factories has become a sustainable and increasingly promising approach for the synthesis of valuable chemicals. However, introducing heterologous pathways into these cell factories can disrupt the endogenous cellular metabolism, leading to suboptimal production performance. To address this challenge, dynamic pathway regulation has been developed and proven effective in improving microbial biosynthesis. In this review, we summarized typical dynamic regulation strategies based on their control logic. The applicable scenarios for each control logic were highlighted and perspectives for future research direction in this area were discussed.

Keywords: Control logic; Dynamic regulation; Feedback control; Oscillation.

Figure 1.. Schemes of two-phase dynamic regulation.

(a) Chemical inducer-triggered two-phase dynamic regulation. The regulators occupy the −10 and −35 regions and block the access of RNA polymerase. The addition of specific chemical inducers will result in a conformational change of the regulator, making it unable to bind the DNA sequence and thus RNA polymerase will bind and start transcription. (b) Temperature-triggered two-phase dynamic regulation. Using the PR/PL-CI system as an example, the promoter PR/PL is repressed by a thermosensitive transcriptional regulator CI dimmer at 30 °C. Increasing the temperature to 37 °C results in a conformational change and the CI monomer will release the repression on promoter PR/PL. (c) Light triggered-two phase dynamic regulation. In the darkness, the promoter cannot work normally without the binding of VP16-EL222 complex. The conformational change of VP16-EL222 complex that is triggered by 425 nm blue light can activate the promoter C120. VP16-EL222: a fusion of EL222 with the transcriptional activation domain of VP16 and a nuclear localization signal; HTH: helix-turn-helix DNA-binding domain. LOV: Light-oxygen-voltage.

Figure 2.. Schemes of positive feedback control-based autonomous dynamic regulation.

(a) Cellular metabolite-based positive feedback control. With cell growth, the increased concentration of the specific metabolite will strengthen the output of the control valve, together with strengthening related gene activation or repression to promote production, forming a positive feedback control-based dynamic regulation. (b) Final product-triggered feedback control. The genes that are responsible for product biosynthesis and genes that compete with heterologous pathways are under the control of the product-triggered promoter. With the accumulation of the final product, the related genes will be activated or repressed autonomously. (c) The mechanism of QS system. The AHL (3-oxohexanoylhomoserine lactone) synthesized by AHL synthase can release the repression by a related repressor, and the promoter can work normally. (d) QS system-triggered positive feedback control. Key genes are under the control of a QS-triggered promoter. With the accumulation of AHL, the genes can be activated or repressed autonomously.

Figure 3.. Schemes of oscillation-based autonomous dynamic regulation.

The gradual accumulation of a specific intermediate will trigger the downstream consumption pathway for production as well as repression of the upstream formation pathway and competing pathway. With the intermediate consumption for production, decreased concentration of intermediate will turn off downstream consuming genes, and at the same time turn on upstream genes for intermediate formation, forming an oscillated gene activation and repression.

Figure 4.. Schemes of multi-functional autonomous dynamic regulation.

More than one biosensor and control logic were used to coordinate gene expression and repression. As shown in the figure, this multi-functional autonomous dynamic regulation network includes intermediate-triggered oscillation and final product-triggered positive feedback control.