Cell-free (in vitro) protein synthesis (CFPS) systems provide a
versatile tool that can be used to investigate different aspects of the
transcription-translation machinery by reducing cells to the basic
functions of protein formation. Recent improvements in reaction
stability and lysate preparation offer the potential to expand the scope
of in vitro biosynthesis from a research tool to a multifunctional and
versatile platform for protein production and synthetic biology. To
date, even the best-performing CFPS systems are drastically slower than
in vivo references. Major limitations are imposed by ribosomal
activities that progress in an order of magnitude slower on the mRNA
template. Owing to the complex nature of the ribosomal machinery,
conventional ``trial and error'' experiments only provide little
insight into how the desired performance could be improved. By applying
a DNA-sequence-oriented mechanistic model, we analyzed the major
differences between cell-free in vitro and in vivo protein synthesis. We
successfully identified major limiting elements of in vitro translation,
namely the supply of ternary complexes consisting of EFTu and tRNA.
Additionally, we showed that diluted in vitro systems suffer from
reduced ribosome numbers. On the basis of our model, we propose a new
experimental design predicting 90\% increased translation rates, which
were well achieved in experiments. Furthermore, we identified a shifting
control in the translation rate, which is characterized by availability
of the ternary complex under in vitro conditions and the initiation of
translation in a living cell. Accordingly, the model can successfully be
applied to sensitivity analyses and experimental design.
We thank Eike Krauter and Michael Kraml for their assistance with the
cell-free reactions. We further gratefully acknowledge the funding of
this work by the Bundesministerium fur Bildung and Forschung (BMBF;
Grant FKZ031A157D) and the European Union (''ST-Flow'' project,
Grant 289326 in the framework 7 program KBBE.2011.3.6-03).
%0 Journal Article
%1 ISI:000413715200013
%A Niess, Alexander
%A Failmezger, Jurek
%A Kuschel, Maike
%A Siemann-Herzberg, Martin
%A Takors, Ralf
%C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA
%D 2017
%I AMER CHEMICAL SOC
%J ACS SYNTHETIC BIOLOGY
%K myown proteinsynthesis
%N 10
%P 1913-1921
%R 10.1021/acssynbio.7b00117
%T Experimentally Validated Model Enables Debottlenecking of in Vitro
Protein Synthesis and Identifies a Control Shift under in Vivo
Conditions
%U https://doi.org/10.1021/acssynbio.7b00117
%V 6
%X Cell-free (in vitro) protein synthesis (CFPS) systems provide a
versatile tool that can be used to investigate different aspects of the
transcription-translation machinery by reducing cells to the basic
functions of protein formation. Recent improvements in reaction
stability and lysate preparation offer the potential to expand the scope
of in vitro biosynthesis from a research tool to a multifunctional and
versatile platform for protein production and synthetic biology. To
date, even the best-performing CFPS systems are drastically slower than
in vivo references. Major limitations are imposed by ribosomal
activities that progress in an order of magnitude slower on the mRNA
template. Owing to the complex nature of the ribosomal machinery,
conventional ``trial and error'' experiments only provide little
insight into how the desired performance could be improved. By applying
a DNA-sequence-oriented mechanistic model, we analyzed the major
differences between cell-free in vitro and in vivo protein synthesis. We
successfully identified major limiting elements of in vitro translation,
namely the supply of ternary complexes consisting of EFTu and tRNA.
Additionally, we showed that diluted in vitro systems suffer from
reduced ribosome numbers. On the basis of our model, we propose a new
experimental design predicting 90\% increased translation rates, which
were well achieved in experiments. Furthermore, we identified a shifting
control in the translation rate, which is characterized by availability
of the ternary complex under in vitro conditions and the initiation of
translation in a living cell. Accordingly, the model can successfully be
applied to sensitivity analyses and experimental design.
@article{ISI:000413715200013,
abstract = {{Cell-free (in vitro) protein synthesis (CFPS) systems provide a
versatile tool that can be used to investigate different aspects of the
transcription-translation machinery by reducing cells to the basic
functions of protein formation. Recent improvements in reaction
stability and lysate preparation offer the potential to expand the scope
of in vitro biosynthesis from a research tool to a multifunctional and
versatile platform for protein production and synthetic biology. To
date, even the best-performing CFPS systems are drastically slower than
in vivo references. Major limitations are imposed by ribosomal
activities that progress in an order of magnitude slower on the mRNA
template. Owing to the complex nature of the ribosomal machinery,
conventional ``trial and error{''} experiments only provide little
insight into how the desired performance could be improved. By applying
a DNA-sequence-oriented mechanistic model, we analyzed the major
differences between cell-free in vitro and in vivo protein synthesis. We
successfully identified major limiting elements of in vitro translation,
namely the supply of ternary complexes consisting of EFTu and tRNA.
Additionally, we showed that diluted in vitro systems suffer from
reduced ribosome numbers. On the basis of our model, we propose a new
experimental design predicting 90\% increased translation rates, which
were well achieved in experiments. Furthermore, we identified a shifting
control in the translation rate, which is characterized by availability
of the ternary complex under in vitro conditions and the initiation of
translation in a living cell. Accordingly, the model can successfully be
applied to sensitivity analyses and experimental design.}},
added-at = {2018-01-25T13:38:08.000+0100},
address = {{1155 16TH ST, NW, WASHINGTON, DC 20036 USA}},
affiliation = {{Takors, R (Reprint Author), Univ Stuttgart, Inst Biochem Engn, D-70569 Stuttgart, Germany.
Niess, Alexander; Failmezger, Jurek; Kuschel, Maike; Siemann-Herzberg, Martin; Takors, Ralf, Univ Stuttgart, Inst Biochem Engn, D-70569 Stuttgart, Germany.}},
author = {Niess, Alexander and Failmezger, Jurek and Kuschel, Maike and Siemann-Herzberg, Martin and Takors, Ralf},
author-email = {{takors@ibvt.uni-stuttgart.de}},
biburl = {https://puma.ub.uni-stuttgart.de/bibtex/283d4527cc21beb75d30e799b1d77d87c/siemannherzberg},
da = {{2018-01-25}},
doc-delivery-number = {{FK7VK}},
doi = {{10.1021/acssynbio.7b00117}},
funding-acknowledgement = {{Bundesministerium fur Bildung and Forschung (BMBF) {[}FKZ031A157];
European Union {[}289326, KBBE.2011.3.6-03]}},
funding-text = {{We thank Eike Krauter and Michael Kraml for their assistance with the
cell-free reactions. We further gratefully acknowledge the funding of
this work by the Bundesministerium fur Bildung and Forschung (BMBF;
Grant FKZ031A157D) and the European Union ({''}ST-Flow{''} project,
Grant 289326 in the framework 7 program KBBE.2011.3.6-03).}},
interhash = {ea124a9a8adf05f27614b52291b8d790},
intrahash = {83d4527cc21beb75d30e799b1d77d87c},
issn = {{2161-5063}},
journal = {{ACS SYNTHETIC BIOLOGY}},
journal-iso = {{ACS Synth. Biol.}},
keywords = {myown proteinsynthesis},
keywords-plus = {{CELL-FREE SYSTEM; ESCHERICHIA-COLI; TRANSLATION INITIATION;
GENE-EXPRESSION; MESSENGER-RNA; TRANSCRIPTION; BACTERIA; SUBUNITS;
GLUCOSE; BINDING}},
language = {{English}},
month = {{OCT}},
number = {{10}},
number-of-cited-references = {{38}},
pages = {{1913-1921}},
publisher = {{AMER CHEMICAL SOC}},
research-areas = {{Biochemistry \& Molecular Biology}},
times-cited = {{1}},
timestamp = {2018-06-14T11:13:05.000+0200},
title = {{Experimentally Validated Model Enables Debottlenecking of in Vitro
Protein Synthesis and Identifies a Control Shift under in Vivo
Conditions}},
type = {{Article}},
unique-id = {{ISI:000413715200013}},
url = {https://doi.org/10.1021/acssynbio.7b00117},
usage-count-last-180-days = {{2}},
usage-count-since-2013 = {{2}},
volume = {{6}},
web-of-science-categories = {{Biochemical Research Methods}},
year = {{2017}}
}