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

Michael C Jewett^1,a, Kara A Calhoun¹, Alexei Voloshin¹, Jessica J Wuu¹ & James R Swartz^1,2

^aPresent address: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA

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Synopsis

Cell-free systems provide a valuable platform for understanding, using, and expanding the capabilities of natural systems (Forster and Church, 2006, 2007; Swartz, 2006; Doktycz and Simpson, 2007; Meyer et al, 2007). For example, they played an important role in deciphering the genetic code (Nirenberg, 2004). More recently, cell-free systems have been used as a test bed for understanding and building genetic circuits (Noireaux et al, 2003; Kim et al, 2006) and have shown remarkable utility as a protein synthesis technology (Katzen et al, 2005; Swartz, 2006), including the production of patient-specific vaccine candidates (Yang et al, 2005; Kanter et al, 2007) and pharmaceutical proteins (Yang et al, 2005; Goerke and Swartz, 2008).

Despite being used for decades as a tool in fundamental and applied research, one major disadvantage of most cell-free systems is their inability to co-activate multiple complex biochemical networks in a single integrated platform. In this study, we have addressed this challenge. By quantitatively assessing active biochemical reactions in an Escherichia coli-based crude extract cell-free protein synthesis (CFPS) system, the Cytomim system (Jewett and Swartz, 2004a), we reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. First, we show that CFPS can be carried out without the addition of a secondary energy substrate (Figure 2), suggesting a novel energy generation mechanism. Second, we discover that glutamate is directed into TCA cycle intermediates and side products (Figure 2). This result led us to hypothesize that inverted inner membrane vesicles (IMVs) competent for oxidative phosphorylation (generated by our high shear rate lysis procedure and retained in the extract) were activated to produce ATP from reducing equivalents generated by catabolism. Consistent with this hypothesis, we show that protein synthesis is indeed significantly stimulated by oxygen availability (Figure 3). Finally, by using biochemical inhibitors of the electron transport chain and the F₁F_O-ATPase as well as membrane gradient uncouplers, we reveal that respiration from IMVs is active and fuels protein synthesis in vitro (Figure 3). The overall picture that emerges from our study is that IMVs serve to convert reducing equivalents gained from glutamate catabolism into the ATP required to fuel high-level transcription and translation. Never before have this many complex enzyme systems been shown to be simultaneously activated without living cells.

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 l 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 l 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 l 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.

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Figure 3

Oxygen-dependent energy production in the Cytomim system is caused by oxidative phosphorylation. (A) Here, 20 l 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, 144764 mg/l of CAT is synthesized when oxygen is present. In (C), 20 l 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 M 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 l 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 n6 experiments. (B) n=3. Error bars=1 s.d.

Full figure and legend (248K)Figures & Tables index

As this crude extract system can provide integrated metabolic function, we believe that the Cytomim system lays the foundation for constructive cell-free synthetic biology projects. Past cell-free systems have typically lacked the 'housekeeping functions' of the cell (Simpson, 2006), such as activated metabolic networks, sustained energy production, and highly productive protein synthesis.

It is striking to note that the Cytomim system closely mimics E. coli cellular metabolism. It is homeostatic in pH and [P_i], uses natural, non-phosphorylated energy substrates, provides a long-lasting ATP source, and fuels highly productive protein synthesis (up to 600 mg protein/l/h). In addition, each ribosome can polymerize approximately 10 500 amino acids (42 copies of chloramphenicol acetyl transferase, CAT), indicating that the Cytomim system is not limited by enzyme turnover (e.g. only one protein, or fraction of a protein, produced per ribosome). Furthermore, the specific oxygen uptake rate in the Cytomim system is on the same order as for intact E. coli cells.

We also describe a more cost-effective and highly productive cell-free protein production technology. High reagent costs, primarily energy substrates and nucleotides (amino acid costs are relatively minor), and low productivities have been a major deterrent for the commercial use of CFPS systems (Swartz, 2006). We show that supplementing low concentrations of phosphate increases cell-free protein production in the Cytomim/glutamate system and also that NMPs are effective nucleotides for the Cytomim/glutamate-phosphate system. The new Cytomim/glutamate-phosphate/NMP batch system is high yielding (up to 1200 mg CAT/l in 2 h; Figure 3B) and more than two orders of magnitude more productive, on a cost basis, than conventional CFPS systems. This advance opens the door for commercial production of personalized medicines and for rapid synthesis of large quantities of vaccines and antidotes against bio-warfare attacks using cell-free factories.

In summary, although in vitro translation systems have been utilized for more than 50 years, demonstration that central metabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a well-defined, robust, and customizable system is new. Given the capability to modify and control cell-free systems, the Cytomim platform will be a powerful tool for systems biology, for synthetic biology, and as a protein production technology.

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