Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

Dudley, Quentin M.; Nash, Connor J.; Jewett, Michael C.

doi:10.1093/synbio/ysz003

Title: Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

Journal Article · 14 January 2019 · Synthetic Biology

DOI: https://doi.org/10.1093/synbio/ysz003 · OSTI ID:1496316

Dudley, Quentin M. ^[1]; Nash, Connor J. ^[1]; Jewett, Michael C. ^[2]

Northwestern Univ., Evanston, IL (United States)
Northwestern Univ., Evanston, IL (United States). Chemistry of Life Processes Inst., Center for Synthetic Biology, and Robert H. Lurie Comprehensive Cancer; Northwestern Univ., Chicago, IL (United States). Simpson Querrey Inst.

Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD⁺, ATP and CoA, the system can synthesize 0.66 mM (90.2 mg l^-1) limonene over 24 h, a productivity of 3.8 mg l^-1 h^-1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.

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Research Organization:: Northwestern Univ., Evanston, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; David and Lucile Packard Foundation; Dreyfus Teacher-Scholar Program; National Institutes of Health (NIH); Dreyfus

Grant/Contract Number:: SC0018249; 503280

OSTI ID:: 1496316

Alternate ID(s):: OSTI ID: 1660452

Journal Information:: Synthetic Biology, Vol. 4, Issue 1; ISSN 2397-7000

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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