CFPS systems have many advantages as a complement to existing protein production technologies for producing disease treatments and diagnostics that are difficult to make in vivo
. This includes therapeutic proteins that (i) are toxic to cells, (ii) require in vitro
selections, (iii) require sequential production, or (iv) require rapid product/process development (e.g. personalized medicines). CFPS methods, however, have been limited by their inability to supply the intense energy needs of protein synthesis without deleterious changes to the chemical environment. This limitation was convincingly demonstrated in the landmark experiments of Spirin et al (1988)
when they dramatically enhanced product yields by continually feeding the energy substrates. Yet, two decades have elapsed without the development of a simple, durable, and cost-effective batch cell-free system. More broadly, integrating complex metabolic networks is an outstanding challenge in cell-free synthetic biology (Simpson, 2006
Here, by evaluating biochemical reactions, we determine the new source of ATP production in the Cytomim CFPS system (Jewett and Swartz, 2004a
). Remarkably, we reveal that central metabolism, oxidative phosphorylation, protein synthesis, and protein folding are simultaneously activated in a single integrated platform. The two intense centrifugation steps in the cell extract preparation procedure remove any intact cells remaining after the high-pressure homogenization step, but IMVs do not sediment. IMVs serve to convert reducing equivalents gained from glutamate catabolism and the TCA cycle 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. Notably, the anionic salt species glutamate is, for the first time, used as a secondary energy substrate. With respect to CFPS systems, this possibility was not previously appreciated and stems from a major frame of reference shift. As our analysis has shown, crude extract systems can now be thought of as a complex set of biochemical reactions that can be identified and controlled, rather than a complex, inscrutable ‘black box'.
It is striking to note that the Cytomim system closely mimics E. coli
cellular metabolism. It is homeostatic in pH and [Pi
], 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 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 intact E. coli
cells. As an additional indication of robust cell-free metabolism, other experiments in our laboratory have shown that the CFPS system exhibits second-order kinetics, i.e. when we doubled the cell extract concentration by ultrafiltration during the course of protein synthesis, volumetric protein synthesis rates increased four-fold (Jewett MC and Swartz JR (2008), in preparation). In other words, the specific translation rate is proportional to the cell extract (ribosome and cofactors) concentration. Cell-free protein elongation rates determined using polysome analysis were consistent with this interpretation (Underwood et al, 2005
). Therefore, the maximum batch protein synthesis rate we observe for the Cytomim system suggests that this complex catalytic system is functioning at approximately the rate expected after a 20-fold dilution from cytoplasmic concentrations. To put this in perspective, even though the cell-free specific elongation rates are lower, the cell-free volumetric productivity compares favorably to typical rates for live cell cultures since the cell-free system uses the entire reactor volume rather the much smaller intracellular volume fraction used in typical cultures.
As our 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. Building synthetic systems of the complexity shown here from the ground up (Forster and Church, 2006
) may ultimately be a more powerful approach for designing synthetic systems. However, the Cytomim platform represents an economically viable platform that can be used now for understanding and using biology, while gaps in our ability to construct purified systems are filled. For example, the Cytomim platform may also be combined with vesicle bioreactors (Noireaux and Libchaber, 2004
; Murtas et al, 2007
) as a further step toward making an artificial cell.
Beyond construction efforts, the Cytomim system also lays the foundation for analyzing complex biomolecular systems that are difficult to manipulate in vivo
without the risk of new enzyme synthesis in response to changing environments. For example, this system will allow a more careful examination of the effects of component localization on overall system performance. It has already enabled us to determine the dependence of protein synthesis rate on [ATP] and [GTP] (Jewett et al, 2008
) and is expected to stimulate intriguing hypotheses that can then be tested in vivo
. For example, our metabolic flux measurements support the concept that many bacteria use intracellular glutamate as an emergency energy source under aerobic conditions following external energy source exhaustion.
Here, we have also engineered a more cost-effective, highly productive Cytomim system. 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 cell-free systems (Swartz, 2006
). The new Cytomim/glutamate-phosphate/NMP batch system is more than two orders of magnitude more productive, on a cost basis, than the conventional PANOx/PEP/NTP system (). This high-yielding (up to 1200 mg/l in 2 h), cost-effective protein synthesis technology is enabling us to produce complex pharmaceutical proteins, such as human insulin-like growth factor-1 at the 1-L scale (Voloshin and Swartz, 2007
). 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.
Activating central metabolism in cell-free extracts also has utility for cofactor-dependent, multistep bioconversions. We have shown, for example, that the Cytomim system can convert inexpensive substrates, such as glutamate, to high-value products such as succinate (Ygs
=0.68 C-mol succinate C-mol glutamate−1
) () (Werpy and Petersen, 2004
). More precise engineering of these pathways should lead to economically attractive bioconversions (Liao, 2004
The reason for protein synthesis termination in the Cytomim system remains an unanswered question. As small molecular weight substrates appear to be in sufficient concentrations (; Supplementary Figure 1
; Supplementary Table II
), inhibitory by-product accumulation or system degradation may be responsible.
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.