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

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.

[1]  M. Jewett,et al.  Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm , 2004, Biotechnology and bioengineering.

[2]  Michael C. Jewett,et al.  Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation , 2008, Journal of bacteriology.

[3]  S. McLaughlin.  The mechanism of action of DNP on phospholipid bilayer membranes , 1972, The Journal of Membrane Biology.

[4]  M. J. Teixeira De Mattos,et al.  Growth yield and energy distribution , 1996 .

[5]  X. Xie,et al.  Living Cells as Test Tubes , 2006, Science.

[6]  Geoffrey Chang,et al.  The past, present and future of cell-free protein synthesis. , 2005, Trends in biotechnology.

[7]  Michael L Simpson,et al.  Cell-free synthetic biology: a bottom-up approach to discovery by design , 2006, Molecular systems biology.

[8]  Kim A Woodrow,et al.  Cell-free protein synthesis with prokaryotic combined transcription-translation. , 2004, Methods in molecular biology.

[9]  J.,et al.  The Purification and Characterization of the Cytochrome d Terminal Oxidase Complex of the Escherichia coli Aerobic Respiratory Chain * , 2022 .

[10]  J. Swartz,et al.  An Economical Method for Cell‐Free Protein Synthesis using Glucose and Nucleoside Monophosphates , 2008, Biotechnology progress.

[11]  M. Jewett,et al.  Mimicking the Escherichia coli cytoplasmic environment activates long‐lived and efficient cell‐free protein synthesis , 2004, Biotechnology and bioengineering.

[12]  Johnathan E. Holladay,et al.  Top Value Added Chemicals From Biomass. Volume 1 - Results of Screening for Potential Candidates From Sugars and Synthesis Gas , 2004 .

[13]  Jim Swartz,et al.  Developing cell-free biology for industrial applications , 2006, Journal of Industrial Microbiology and Biotechnology.

[14]  J. Swartz,et al.  High yield cell-free production of integral membrane proteins without refolding or detergents. , 2008, Biochimica et biophysica acta.

[15]  J. Swartz,et al.  Development of cell‐free protein synthesis platforms for disulfide bonded proteins , 2008, Biotechnology and bioengineering.

[16]  George M Church,et al.  Synthetic biology projects in vitro. , 2006, Genome research.

[17]  P. C. Hinkle,et al.  Oxidative phosphorylation and proton translocation in membrane vesicles prepared from Escherichia coli. , 1974, Biochemical and biophysical research communications.

[18]  Marshall Nirenberg,et al.  Historical review: Deciphering the genetic code--a personal account. , 2004, Trends in biochemical sciences.

[19]  Y. Moriyama,et al.  One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters. , 1991, The Journal of biological chemistry.

[20]  Sven Panke,et al.  Bioengineering novel in vitro metabolic pathways using synthetic biology. , 2007, Current opinion in microbiology.

[21]  M. Jewett,et al.  Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell‐Free Protein Synthesis , 2008, Biotechnology progress.

[22]  A. Spirin,et al.  A continuous cell-free translation system capable of producing polypeptides in high yield. , 1988, Science.

[23]  Takuya Ueda,et al.  Cell-free translation reconstituted with purified components , 2001, Nature Biotechnology.

[24]  M. L. Simpson,et al.  Nano-enabled synthetic biology , 2007, Molecular systems biology.

[25]  J. Hare.  Purification and characterization of a dicyclohexylcarbodiimide-sensitive adenosine triphosphatase complex from membranes of Escherichia coli. , 1975, Biochemical and biophysical research communications.

[26]  George M Church,et al.  Towards synthesis of a minimal cell , 2006, Molecular systems biology.

[27]  J. Swartz,et al.  Energizing cell-free protein synthesis with glucose metabolism. , 2005, Biotechnology and bioengineering.

[28]  J. Hamilton,et al.  The function of ubiquinone in Escherichia coli. , 1970, The Biochemical journal.

[29]  W. Prichard,et al.  A new class of uncoupling agents--carbonyl cyanide phenylhydrazones. , 1962, Biochemical and biophysical research communications.

[30]  Marshall W. Nirenberg,et al.  The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides , 1961, Proceedings of the National Academy of Sciences.

[31]  James Swartz,et al.  Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome. , 2004, Metabolic engineering.

[32]  Dong-Myung Kim,et al.  Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. , 2001, Biotechnology and bioengineering.

[33]  Custom design of metabolism , 2004, Nature Biotechnology.

[34]  David S. Latchman,et al.  Biochemistry (4th edn) , 1995 .

[35]  R. Bar-Ziv,et al.  Principles of cell-free genetic circuit assembly , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[36]  A. Tappel.  Inhibition of electron transport by antimycin A, alkyl hydroxy naphthoquinones and metal coordination compounds. , 1960, Biochemical pharmacology.

[37]  Junhao Yang,et al.  Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines. , 2007, Blood.

[38]  Junhao Yang,et al.  Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system. , 2005, Biotechnology and bioengineering.

[39]  Vincent Noireaux,et al.  A vesicle bioreactor as a step toward an artificial cell assembly. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[40]  A. Yamamoto,et al.  Subunit rotation of ATP synthase embedded in membranes: a or β subunit rotation relative to the c subunit ring , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[41]  E. Winfree,et al.  Construction of an in vitro bistable circuit from synthetic transcriptional switches , 2006, Molecular systems biology.

[42]  Joseph D Puglisi,et al.  Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis. , 2005, Biotechnology and bioengineering.

[43]  Alberto Diaspro,et al.  Protein synthesis in liposomes with a minimal set of enzymes. , 2007, Biochemical and biophysical research communications.