In recent years, driven by innovations in cutting-edge biotechnology and the continuous development of the industrial biotechnology industry, the role and advantages of dynamic regulation strategies have become increasingly prominent in the field of metabolic engineering. To timely balance the relationship between gene expression required for product synthesis and global metabolic levels, the introduction of dynamically regulated genetic pathways enables real-time response to metabolic signals and timely feedback regulation, so as to adapt to changes in the host’s internal metabolism or external environment, achieve stable synthesis of target products, and improve the stability and scalability of fermentation culture.
Generally, dynamic regulation is realized by designing and constructing genetic pathway modules, which allows recombinant cells to dynamically sense and process specific signals, and thus perform adaptive regulation according to signal changes to achieve the optimal state of product synthesis or target systems. The specific signals in this system can be factors such as intermediates in metabolic pathways, metabolites or proteins of the cellular system, as well as signal molecules synthesized by exogenous introduction.
Compared with static regulation, dynamic regulation is a signal-responsive dynamic process that enables online sensing of cellular status and real-time metabolic regulation. This strategy has been applied in numerous studies and achieved a substantial increase in the yield of target products, such as high-value-added biosynthetic products including lycopene, fatty acids, amino acids and inositol. Dynamic regulation systems can be divided into metabolite-dependent dynamic regulation and non-metabolite-dependent dynamic regulation based on different construction principles or response mechanisms; and into unidirectional dynamic regulation and bidirectional dynamic regulation based on different regulatory modes.
Metabolite-dependent dynamic regulation refers to a regulatory mode that takes the metabolites of the target pathway and the endogenous metabolites altered by the introduction of the target pathway as response signals. In contrast, non-metabolite-dependent dynamic regulation uses external environmental factors or exogenously introduced regulatory factors orthogonal to the cellular system as response signals.
Unidirectional dynamic regulation only performs a single up-regulation or down-regulation of target gene expression based on response signals, while bidirectional dynamic regulation can achieve simultaneous up-regulation and down-regulation.
2.1 Metabolite-dependent Dynamic Regulation
Metabolite-dependent dynamic regulation is usually achieved by constructing biosensors to sense metabolite concentrations, and the output signals of these sensors can directly or indirectly regulate the expression of target genes. The efficient synthesis of lycopene is a typical research case. Excessive glucose supply during the growth of Escherichia coli leads to the accumulation of acetic acid, which not only inhibits cell growth but also reduces the metabolic flux of the lycopene synthesis pathway, thereby decreasing the lycopene yield in Escherichia coli.
Studies have found that phosphoenolpyruvate synthase (Pps) is the key to balancing intracellular glyceraldehyde-3-phosphate and pyruvate, while isopentenyl pyrophosphate isomerase (Idi) is the rate-limiting step in the lycopene synthesis pathway. In the glycolysis pathway, acetyl phosphate is an intermediate product generated from pyruvate to acetic acid; therefore, constructing a biosensor with acetyl phosphate molecules as the response signal is the key to realizing the dynamic regulation of the lycopene synthesis pathway.
To this end, researchers have constructed a dynamic regulatory switch responsive to acetyl phosphate concentration. When abnormal accumulation of intracellular acetyl phosphate occurs, the expression levels of phosphoenolpyruvate synthase (Pps) and isopentenyl pyrophosphate isomerase (Idi) are up-regulated, thereby converting pyruvate back to phosphoenolpyruvate, an upstream synthetic precursor of lycopene. Meanwhile, the overexpression of isopentenyl pyrophosphate isomerase (Idi) increases the expression of key enzymes in lycopene synthesis and enhances the metabolic flux of lycopene, thus achieving the goals of reducing acetic acid accumulation and boosting lycopene synthesis. Comparative studies have shown that the titrated lycopene yield obtained by dynamic regulation is 18 times higher than that by simple gene overexpression, with a significant improvement in productivity as well.
2.2 Non-metabolite-dependent Dynamic Regulation
Non-metabolite-dependent dynamic regulation achieves dynamic regulation of gene expression by sensing external environmental factors or signal molecules orthogonal to the host cell system. Compared with metabolite-dependent dynamic regulation, the key feature of this regulatory mode is its independence from the target metabolic pathway and strong universality, but it is necessary to focus on evaluating the impact of exogenous signal introduction on host metabolism. At present, this type of dynamic regulation mainly includes quorum sensing, light control, non-coding RNA, temperature control and other strategies.
Quorum sensing is a typical regulatory mode in which specific signal molecules (e.g., lactones) accumulate with cell growth in a cell population, and the accumulated signal molecules can induce or repress the expression of specific genes at a certain cell density, enabling cells to respond to cell density during growth. It has been widely used in the field of metabolic regulation.
The quorum sensing system is used to perform controllable restriction on the metabolic flux directed to endogenous metabolic pathways, so as to increase the metabolic flux of carbon sources into the target product synthesis pathway. Furthermore, it dynamically controls the metabolic flux of glycolysis and product synthesis pathways in Escherichia coli to balance the coordination between cell growth and product synthesis, achieving an optimal product titer. This method has been applied to the synthesis of shikimic acid, glucaric acid, inositol, bisabolene and other products.
2.3 Dynamic Bidirectional Regulation
At present, dynamic bidirectional regulation is mainly used to resolve the contradiction between early cell growth and late product synthesis, realize stage-specific regulation between the target synthesis pathway and the competing growth-dependent metabolic pathways, and maximize cell growth and product accumulation. For example, a dynamic bidirectional regulation system designed by combining a light-controlled dynamic expression system and a NOT gate genetic circuit has achieved high yield of isobutanol in yeast.
In addition, Dinh et al. constructed a cell density-sensing bidirectional dynamic self-regulation system by combining two quorum sensing systems (repressive and activatory) responsive to the same lactone molecule (OC6), which respectively realized the efficient synthesis of naringenin and salicylic acid in recombinant Escherichia coli. Besides, dynamic bidirectional regulation can currently be used to synthesize artificially customized biological macromolecules, such as block protein complexes and polymeric biological polyester materials.
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