Rewiring cell-free metabolic flux in E. coli lysates using a block—push—pull approach

Dinglasan, Jaime Lorenzo N.; Doktycz, Mitchel J.

doi:10.1093/synbio/ysad007

Title: Rewiring cell-free metabolic flux in E. coli lysates using a block—push—pull approach

Journal Article · Mon Apr 17 00:00:00 EDT 2023 · Synthetic Biology

DOI:https://doi.org/10.1093/synbio/ysad007· OSTI ID:1973294

Dinglasan, Jaime Lorenzo N.;

Abstract Cell-free systems can expedite the design and implementation of biomanufacturing processes by bypassing troublesome requirements associated with the use of live cells. In particular, the lack of survival objectives and the open nature of cell-free reactions afford engineering approaches that allow purposeful direction of metabolic flux. The use of lysate-based systems to produce desired small molecules can result in competitive titers and productivities when compared to their cell-based counterparts. However, pathway crosstalk within endogenous lysate metabolism can compromise conversion yields by diverting carbon flow away from desired products. Here, the ‘block—push—pull’ concept of conventional cell-based metabolic engineering was adapted to develop a cell-free approach that efficiently directs carbon flow in lysates from glucose and toward endogenous ethanol synthesis. The approach is readily adaptable, is relatively rapid and allows for the manipulation of central metabolism in cell extracts. In implementing this approach, a block strategy is first optimized, enabling selective enzyme removal from the lysate to the point of eliminating by-product-forming activity while channeling flux through the target pathway. This is complemented with cell-free metabolic engineering methods that manipulate the lysate proteome and reaction environment to push through bottlenecks and pull flux toward ethanol. The approach incorporating these block, push and pull strategies maximized the glucose-to-ethanol conversion in an Escherichia coli lysate that initially had low ethanologenic potential. A 10-fold improvement in the percent yield is demonstrated. To our knowledge, this is the first report of successfully rewiring lysate carbon flux without source strain optimization and completely transforming the consumed input substrate to a desired output product in a lysate-based, cell-free system.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1973294

Alternate ID(s):: OSTI ID: 1999079

Journal Information:: Synthetic Biology, Journal Name: Synthetic Biology Vol. 8 Journal Issue: 1; ISSN 2397-7000

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
lysate-based
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maximum yield
biotransformation
block—push—pull

Title: Rewiring cell-free metabolic flux in E. coli lysates using a block—push—pull approach

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