Biology Reference
In-Depth Information
enzyme-catalyzed biochemical reaction cascades to produce the desired natural product.
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The easily controlled and engineered open cell-free environment will be exploited for
optimizing multienzyme synthesis and activity for next-generation biocatalysts. Efforts such
as those by Forster and colleagues could provide directions.
142,143
Purification and Localization of Multienzyme Ensembles
To produce a desired product from an inexpensive feedstock, multiple biocatalytic steps are
often necessary. A key challenge, therefore, for bottom-up cell-free systems is cost-effective
purification of multienzyme ensembles. Offering one possible solution, a recent report from
Wang et al. leverages advanced genome engineering for simultaneous modification of
catalysts to be used in copurification strategies.
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This type of strategy, once further
developed, could lead to commercially relevant in vitro catalytic systems. Beyond strategies
for simultaneously modifying and copurifying large protein complexes and pathways,
localization may also be important. Nature has evolved highly efficient mechanisms to
facilitate the transfer of reactants through complex enzymatic pathways. For the production
of secondary metabolites, for example, type I polyketide synthetases actively transfer the
reactant through a number of catalytic domains that are locally constrained as part of the
same protein. Biomimetic engineering, the ability to control the proximity of multiple
enzymes, could facilitate the rapid transfer of reactants through the correct sequence of
biocatalytic events. Innovations in the rapidly growing enzyme immobilization field could
help facilitate the development of such biomimetic multienzyme ensembles.
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SUMMARY
Cell-free synthetic biology is a powerful tool for understanding and expanding the basic
biology of living systems. Recent advances in nucleic acid circuitry, protein synthesis, and
small molecule production, amongst others, have heralded a new age of in vitro
bioengineering. The general simplicity and flexibility of in vitro systems serves as an
excellent complement to in vivo studies, and enables a more complete understanding of the
inner workings of a cell. Cell-free synthetic biology has versatile applications in protein
synthesis and evolution, as well as the development of nucleic acid circuits, nanomachines,
and therapeutics.
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Although cell-free systems have been used for over 50 years, more work is needed before
they can be used as commercial platforms for therapeutic and metabolite production.
However, recent advances have demonstrated commercial-scale protein synthesis with crude
extract cell-free systems. In addition, continued engineering has rapidly driven down the
cost and simplified the preparation of many cell-free systems. With the remarkable progress
made over the last 10 years, it is but a matter of time before several of the significant
challenges facing the field evolve into transformative synthetic biology opportunities.
Acknowledgments
MCJ and AR gratefully acknowledge funding from the National Institutes of Health (Grant Number
R00GM081450), the National Academies Keck Futures Initiative (Grant Number NAFKI-SB5), the National Science
Foundation (Grant Number MCB-0943393), the Office of Naval Research (Grant Number N00014-11-1-0363), and
the DARPA YFA Program (Grant Number N66001-11-1-4137). BCB and JCW thankfully acknowledge funding from
the National Science Foundation (Award Number 1115229), the Rocky Mountain NASA Space Grant Consortium,
and Utah NASA EPSCoR.
References
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2. Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, et al. Microscale to manufacturing scale-up of cell-free
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