Cell-free protein synthesis: the state of the art

Whittaker, James W.

doi:10.1007/s10529-012-1075-4

Cited by 48 publications

(41 citation statements)

References 68 publications

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“…the PURE system) [39] and commercial applications have been established [40, 41]. CFPS will not be discussed further in this paper, but several other outstanding reviews are available [42–44].…”

Section: Introductionmentioning

confidence: 99%

Cell‐free metabolic engineering: Biomanufacturing beyond the cell

Dudley

Karim

Jewett

2014

Biotechnology Journal

292

258

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Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L−1 hr−1, reaction scales of >100L, and new directions in protein purification, spatial organization and enzyme stability. In the coming years, CFME will offer exciting opportunities to (i) debug and optimize biosynthetic pathways, (ii) carry out design-build-test iterations without re-engineering organisms, and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility.

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Section: Introductionmentioning

confidence: 99%

Cell‐free metabolic engineering: Biomanufacturing beyond the cell

Dudley

Karim

Jewett

2014

Biotechnology Journal

292

258

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“…The PURE is composed of purified recombinant elements necessary for transcribing and translating a coding DNA or RNA sequence, totally in vitro. Briefly, the PURE system includes T7 RNA polymerase, an energy-coupling module for NTP regeneration, transfer RNAs, ribosomes, translation initiation, elongation and release factors in a suitable buffer ( 28, 29 ). By its intrinsic flexibility, a TX-TL system allows us to precisely tune the relative concentrations of the two components of a riboregulator at the DNA and at the RNA level.…”

Section: Resultsmentioning

confidence: 99%

Quantitative characterization of translational riboregulators using an in vitro transcription-translation system

Senoussi

Wah

Shimizu

et al. 2018

Preprint

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2Riboregulators are short RNA sequences that, upon binding to a ligand, change 3 their secondary structure and influence the expression rate of a downstream gene. 4 They constitute an attractive alternative to transcription factors for building synthetic 5 gene regulatory networks because they can be engineered de novo and they have a fast 6 turnover and a low metabolic burden. However, riboregulators are generally designed in 7 silico and tested in vivo, which only provides a yes/no evaluation of their performances, 8 thus hindering the improvement of design algorithms. Here we show that a cell-free 9 transcription-translation (TX-TL) system provides valuable quantitative information 10 about the performances of in silico designed riboregulators. In particular, we use the 11 ribosome as an exquisite molecular machine that detects functional riboregulators, pre-12 cisely measures their concentration and linearly amplifies the signal by generating a 13 fluorescent protein. We apply this method to characterize two types of translational 14 riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At 15 the DNA level we demonstrate that high concentrations of taDNA poisoned the acti-16 vator until total shut off. At the RNA level, we show that this approach provides a 17 fast and simple way to measure dissociation constants of functional riboregulators, in 18 contrast to standard mobility-shift assays. Our method opens the route for using cell-19 free TX-TL systems for the quantitative characterization of functional riboregulators 20 in order to improve their design in silico. 21Keywords 22 in vitro synthetic biology, RNA translational riboregulator, cell-free protein synthesis 23 2 During the early wave of synthetic biology (1 , 2 ), known transcription factors were wired 25 to their corresponding promoter sequences to control the expression of other transcription 26 factors or effector proteins. While this approach has been very successful in engineering 27 gene regulatory networks (GRNs) (3 ) with few nodes, the number of different elements in 28 synthetic GRNs has stagnated at 5-6 (4 ). Two arguments may explain this limit. First, 29 protein-DNA interactions are very difficult to design, although very promising computation 30 methods are arising (5 ); the engineer must thus choose well-known transcription factor-31 promoter pairs. Second, the expression of these transcription factors imposes a metabolic 32 burden to the cells (6 ). 33Implementing regulatory circuits at the RNA level may help solving these issues because 34 RNA-RNA interactions can be predicted from the sequence (7 -9 ) and protein expression is 35 not needed for regulation, which lowers the metabolic burden. The principal component of 36 an RNA-regulated GRN is the riboregulator: an RNA sequence in the 5' untranslated region 37 (UTR) of a gene of interest that has an effect on its expression rate. Since they were first used 38 in synthetic biology more than a decade ago (10 ), several riboregulators have been desi...

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“…However, new products that encourage entry into the field while also enhancing the quality of libraries for established protein engineers are emerging. For example, the PURE system [3,39] is a fully defined IVTT system with a high concentration of ribosomes and low levels of detrimental nucleases and proteases, thus yielding more complex libraries of display particles. Additionally, streamlined protocols are being developed to simplify cell-free evolution [40*,41].…”

Section: Methodological Advances That Have Improved Accessibility Efmentioning

confidence: 99%

Conceptual and methodological advances in cell-free directed evolution

Dodevski

Markou

Sarkar

2015

Current Opinion in Structural Biology

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Although cell-free directed evolution methods have been used to engineer proteins for nearly two decades, selections on more complex phenotypes have largely remained in the domain of cell-based engineering approaches. Here, we review recent conceptual advances that now enable in vitro display of multimeric proteins, integral membrane proteins, and proteins with an expanded amino acid repertoire. Additionally, we discuss methodological improvements that have enhanced the accessibility, efficiency, and robustness of cell-free approaches. Coupling these advances with the in vitro advantages of creating exceptionally large libraries and precisely controlling all experimental conditions, cell-free directed evolution is poised to contribute significantly to our understanding and engineering of more complex protein phenotypes.

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Cell-free protein synthesis: the state of the art

Cited by 48 publications

References 68 publications

Cell‐free metabolic engineering: Biomanufacturing beyond the cell

Cell‐free metabolic engineering: Biomanufacturing beyond the cell

Quantitative characterization of translational riboregulators using an in vitro transcription-translation system

Conceptual and methodological advances in cell-free directed evolution

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