A Synthetic Gene-Metabolic Oscillator
Fung, E., Wong, W.W., Suen, J.K., Bulter, T., Lee, S.G. and Liao, J.C. (2005) A synthetic gene-metabolic oscillator. Nature, 435, 118-122. [Full Text] [PDF]
Autonomous oscillations found in gene expression and metabolic, cardiac and neuronal systems have attracted significant attention both because of their obvious biological roles and their intriguing dynamics. In addition, de novo designed oscillators have been demonstrated, using components that arenot part of the natural oscillators. Such oscillators are useful in testing the design principles and in exploring potential applications not limited by natural cellular behaviour transcriptional and metabolic integration characteristic of natural oscillators, here we designed and constructed a synthetic circuit in Escherichia coli K12, using glycolytic flux to generate oscillation through the signalling metabolite acetyl phosphate. If two metabolite pools are interconverted by two enzymes that are placed under the transcriptional control of acetyl phosphate, the system oscillates when the glycolytic rate exceeds a critical value. We used bifurcation analysis to identify the boundaries of oscillation, and verified these experimentally. This work demonstrates the possibility of using metabolic flux as a control factor in system-wide oscillation, as well as the predictability of a de novo gene–metabolic circuit designed using nonlinear dynamic analysis.
Metabolic Engineering and Gene-Metabolic Circuits
Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to a metabolic imbalance and sub-optimal productivity. We have demonstrated the design and engineering of a regulatory circuit, in addition to amplifying the pathway genes, as the second stage of metabolic engineering effort. In particular, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The altered regulon, stimulated by excess glycolytic flux though sensing of the acetyl phosphate level, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This artificial regulon significantly enhanced lycopene and recombinant protein production and reduced the growth retardation caused by protein overexpression. Projects accomplished are highlighted in the following.
Rewiring metabolic control loop
We have designed an artificial, intracellular feedback control loop that senses an intracellular metabolite as an signal for metabolic flux redirection. See Farmer and Liao (2000) for details.
Carotenoids Production
We have constructed an E. coli strain that are suitable for production of carotenoids from glucose. We have investigated the rate controlling step in the network (Wang et al. 2000), improved the key enzyme using directed evolution (Wang et al. 2001, Wang et al., 2002). In addition, we identified a precursor balancing in carotenoid production (Farmer and Liao, 2001) and constructed an artificial feedback loop for flux control in the recombinant pathway (Farmer and Liao, 2000).
Aromatics production
We were the first group to identify the stoichiometric limitation caused by the phosphotransferase system (PTS) in the production of various metabolites , and experimentally demonstrated a solution by overexpression of phosphoenopyruvate synthase (Pps) to recycle pyruvate back to phosphoenopyruvate (Patnaik and Liao, 1994, Patnaik et al. 1995).
Engineering of Central metabolism
Since central metabolism determines the flow of carbon fluxes, we systematically investigated the role of the key genes involved in pyruvate and phosphoenolpyruvate metabolism (Patnaik et al. 1992, Chao and Liao, 1993, Chao and Liao, 1994, Chao et al. 1994, Hou et al. 1995).
Design of artificial cell-cell communication for gene-metabolic circuits
We demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell-cell communication. This circuit uses a threshold concentration of acetate to induce gene expression via acetate kinase and part of the nitrogen regulation two-component system. As the first application of the cell-cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in E. coli made acetate secretion proportional to cell density and independent of oxygen availability.
Metabolic modeling
Modeling of metabolic and genetic systems has been a long-term interest in our group. We have developed several useful theories and methods for modeling and analysis. These include Dynamic Metabolic Control Theory (Delgado and Liao,1991, 1992, Liao and Delgado, 1992, Delgado et al., 1993), flux analysis using regulatory constraints (Liao and Delgado, 1998), inverse flux analysis (Delgado and Liao, 1997, Liao and Oh, 1999), and metabolic modeling using fuzzy logic models (Yen et al, 1998, Lee et al. 1999). Most importantly, in 1996, we proposed the use of convex analysis (elementary mode analysis) as a way to examine effects of mutation in metabolic systems (Liao et al. 1996).