Title: Elucidating the potential of crude cell extracts for producing pyruvate from glucose

Abstract

Here, living systems possess a rich biochemistry that can be harnessed through metabolic engineering to produce valuable therapeutics, fuels and fine chemicals. In spite of the tools created for this purpose, many organisms tend to be recalcitrant to modification or difficult to optimize. Crude cellular extracts, made by lysis of cells, possess much of the same biochemical capability, but in an easier to manipulate context. Metabolic engineering in crude extracts, or cell-free metabolic engineering, can harness these capabilities to feed heterologous pathways for metabolite production and serve as a platform for pathway optimization. However, the inherent biochemical potential of a crude extract remains ill-defined, and consequently, the use of such extracts can result in inefficient processes and unintended side products. Herein, we show that changes in cell growth conditions lead to changes in the enzymatic activity of crude cell extracts and result in different abilities to produce the central biochemical precursor pyruvate when fed glucose. Proteomic analyses coupled with metabolite measurements uncover the diverse biochemical capabilities of these different crude extract preparations and provide a framework for how analytical measurements can be used to inform and improve crude extract performance. Such informed developments can allow enrichment of crude extracts withmore » pathways that promote or deplete particular metabolic processes and aid in the metabolic engineering of defined products.« less

Authors:

Garcia, David C. ^[1]; Mohr, Benjamin P. ^[1]; Dovgan, Jakob T. ^[2]; Hurst, Gregory B.  ^[3];

• Search DOE PAGES for author "Hurst, Gregory B."
• Search DOE PAGES for ORCID "0000-0002-7650-8009"

Standaert, Robert F. ^[2]; Doktycz, Mitchel J.  ^[1]

• Search DOE PAGES for author "Doktycz, Mitchel J."
• Search DOE PAGES for ORCID "0000-0003-4856-8343"

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+ Show Author Affiliations

1. Bredesen Center for Interdisciplinary Research, University of Tennessee, Knoxville, TN, USA, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA
2. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA
3. Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

Publication Date:

Mon May 14 00:00:00 EDT 2018

Research Org.:

Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1441091

Alternate Identifier(s):

OSTI ID: 1468228

Grant/Contract Number:  

AC05-00OR22725

Resource Type:

Published Article

Journal Name:

Synthetic Biology

Additional Journal Information:

Journal Name: Synthetic Biology Journal Volume: 3 Journal Issue: 1; Journal ID: ISSN 2397-7000

Publisher:

Oxford University Press

Country of Publication:

United Kingdom

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES