Title: Modular cell-free expression plasmids to accelerate biological design in cells

Abstract

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression ofmore » functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.« less

Authors:

Karim, Ashty S.  ^[1];

• Search DOE PAGES for author "Karim, Ashty S."
• Search DOE PAGES for ORCID "0000-0002-5789-7715"

Liew, Fungmin ^[2]; Garg, Shivani ^[3]; Vögeli, Bastian  ^[1];

• Search DOE PAGES for author "Vögeli, Bastian"
• Search DOE PAGES for ORCID "0000-0001-7870-7311"

Rasor, Blake J.  ^[1];

• Search DOE PAGES for author "Rasor, Blake J."
• Search DOE PAGES for ORCID "0000-0001-6662-341X"

Gonnot, Aislinn ^[3]; Pavan, Marilene ^[3]; Juminaga, Alex ^[3]; Simpson, Séan D. ^[3]; Köpke, Michael ^[3]; Jewett, Michael C. ^[1]

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+ Show Author Affiliations

1. Northwestern Univ., Evanston, IL (United States)
2. (Eric) [LanzaTech, Inc., Skokie, IL (United States)
3. LanzaTech, Inc., Skokie, IL (United States)

Publication Date:

Wed Oct 14 00:00:00 EDT 2020

Research Org.:

Northwestern Univ., Evanston, IL (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); Swiss National Science Foundation (SNSF)

OSTI Identifier:

1854008

Alternate Identifier(s):

OSTI ID: 2298969

Grant/Contract Number:  

SC0018249; AC02-05CH11231; P2SKP3_184036

Resource Type:

Accepted Manuscript

Journal Name:

Synthetic Biology

Additional Journal Information:

Journal Volume: 5; Journal Issue: 1; Journal ID: ISSN 2397-7000

Publisher:

Oxford University Press

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; cell-free systems; Clostridium; plasmids; industrial biotechnology; metabolic engineering