An integrated cell-free metabolic platform for protein production and synthetic biology

Abstract

Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.

Figure 1

Diagram of the molecular subsystems shown to be active in the Cytomim cell-free system. Glutamate (GLU) is used as a robust energy source in a natural chemical environment to produce reducing equivalents, primarily in the form of NADH, through the TCA cycle. NADH fuels oxidative phosphorylation in which oxygen serves as the final electron acceptor. Membrane-dependent respiration provides a stable supply of ATP, which is coupled to combined transcription and translation followed by protein folding. To prove the existence of oxidative phosphorylation in the Cytomim cell-free system, inhibitors were used to inactivate the electron transport chain (ETC), dissipate the proton motive force (PMF), and inactivate the F₁F_O-ATPase enzyme complex (each is denoted with an ^*) (αKG, alpha-ketoglutarate; SUC, succinate; MAL, malate; PYR, pyruvate; OAC, acetate; OAA, oxaloacetate; ASP, aspartic acid; P_i, inorganic phosphate; TFs, translation factors; aa-tRNAs, aminoacylated-tRNAs).

Figure 2

Protein product, ATP, and metabolite kinetics for the Cytomim system indicate that central metabolism has been co-activated with protein synthesis. (A) Synthesis of chloramphenicol acetyl transferase (CAT) as determined by ¹⁴C-leucine incorporation in the Cytomim system (15 μl batch reactions) with 33 mM pyruvate (Cytomim/pyruvate/NTP—closed squares), and without pyruvate (Cytomim/glutamate/NTP—open circles). Control transcription and translation reactions without plasmid DNA were used to assess background protein production levels. These reactions demonstrated negligible background incorporation. (B) ATP concentration kinetics during protein synthesis (15 μl batch reactions) confirm the presence of an additional energy substrate besides pyruvate. Cytomim system with 33 mM pyruvate (Cytomim/pyruvate/NTP—closed squares). Cytomim system without pyruvate (Cytomim/glutamate/NTP—open circles). (C) Glutamate (GLU) depletion during protein synthesis (15 μl batch reactions) suggests that glutamate is fueling energy production. Cytomim system with 33 mM pyruvate (Cytomim/pyruvate/NTP—closed squares). Cytomim system without pyruvate (Cytomim/glutamate/NTP—open circles). (D) Organic acid formation kinetics in the Cytomim/pyruvate/NTP system indicate the accumulation of TCA cycle intermediates (OAA, oxaloacetate). As expected, acetate concentrations increased rapidly as a result of pyruvate catabolism through pyruvate dehydrogenase during the first 30 min of the reaction. (E) Organic acid formation kinetics in the Cytomim/glutamate/NTP system indicate the accumulation of TCA cycle intermediates (OAA, oxaloacetate). (F) Accumulation of radioactivity in metabolites of the Cytomim/glutamate/NTP cell-free reaction. A mixture of uniformly labeled ¹⁴C-glutamate and non-labeled glutamate was added at the start of the reaction. (A, B) Results are the average of n=6 experiments. (C–E) n=4. (F) n=3. Error bars=1 s.d.

Figure 3

Oxygen-dependent energy production in the Cytomim system is caused by oxidative phosphorylation. (A) Here, 20 μl cell-free batch reactions were carried out for 5 h. CAT production yields in the Cytomim/pyruvate/NTP system (Pyr) or in the Cytomim/glutamate/NTP system (Glu) in the presence of oxygen (O₂) or argon (Ar). In (B), 2 ml stirred tank cell-free reactions using the Cytomim/glutamate-phosphate/NMP system are shown (see Materials and methods). Total CAT yield (circles), ATP concentration (triangles) and dissolved oxygen concentration (squares) are plotted versus time. After 40 min (see arrow), the oxygen feed was stopped (closed shapes). This resulted in complete consumption of available oxygen, depletion of ATP, and termination of protein synthesis. A control reaction (open shapes) is shown for comparison. After 6 h, 1447±64 mg/l of CAT is synthesized when oxygen is present. In (C), 20 μl cell-free batch reactions carried out for 5 h are shown. CAT production yields in the Cytomim/pyruvate/NTP system (Pyr) or in the Cytomim/glutamate/NTP system (Glu) in the presence of oxygen (O₂) or argon (Ar). The reduction in protein synthesis after addition of either 75 μM 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO, an inhibitor of electron transport), 1 mM thenoyltrifluoroacetone (TTA, an inhibitor of electron transport) or 2.5 mM 2-4-dinitrophenol (DNP, an uncoupling agent) indicates that oxygen-dependent protein synthesis relies on energy derived from oxidative phosphorylation (e.g. Pyr/O₂ versus Pyr/O₂/HQNO). Oxygen-independent CAT synthesis is unaffected (e.g. Pyr/Ar versus Pyr/Ar/HQNO). In (D), 20 μl cell-free batch reactions using the conventional PANOx/PEP/NTP system (not the Cytomim system), carried out for 5 h, are shown. Consistent with previous results (Kim and Swartz, 2001), inhibitors of oxidative phosphorylation do not affect protein biosynthesis in this case (conducted in the presence of oxygen). (A, C, D) Results are the average of n⩾6 experiments. (B) n=3. Error bars=1 s.d.

Figure 4

The Cytomim system provides a chemical environment conducive for oxidative phosphorylation. Quinacrine, a self-quenching fluorescent dye that distributes according to pH, was used to indicate the proton pumping capability of IMVs. (A) When NADH is added to IMVs to initiate proton pumping, a 10% drop in fluorescence signal is observed, consistent with pH-induced quinacrine accumulation inside vesicles. This drop in fluorescence is not seen when vesicles are absent from the solution. As an additional control, the experiment was repeated with vesicles in the presence of 0.1% Triton X-100 detergent (TX100). Triton X-100 dissolves lipid bilayers and is shown here to prevent accumulation of protons or quinacrine. (B) Similar to NADH, we observe a drop in fluorescence when proton pumping through the F₁F_O-ATPase is initiated by ATP addition. (C) Oxygen uptake rate of the Cytomim/glutamate-phosphate/NMP system during synthesis of CAT. Results are the average of n=3 experiments. Error bars=1 s.d. (D) Addition of purified inner membrane vesicles to the Cytomim/glutamate-phosphate/NMP system during synthesis of CAT increases the oxygen uptake rate. At 10 min after the start of the reaction, 0.26 mg of purified inner membrane vesicles were added to the reaction, increasing the total vesicle concentration from 0.26 to 0.36 mg/ml. Results are the average of n=3 experiments. Errors bars=1 s.d.

Figure 5

CAT synthesis over time using the Cytomim/glutamate-phosphate/NTP system. Reactions were carried out for 10 h at 37°C and CAT synthesis was determined by ¹⁴C-leucine incorporation and enzymatic activity assay. Here, 10 mM phosphate was supplemented to the cell-free reaction without pyruvate and 15 μl reaction mixtures were prepared in a different tube for each time point. Results are the average of n=6 experiments. Error bars=1 s.d. Total yield of CAT as determined by ¹⁴C-leucine incorporation (closed squares). Soluble yield of CAT as determined by ¹⁴C-leucine incorporation (gray circles). Active yield of CAT as determined by enzymatic assay (open triangles).