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Title: Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production

Abstract

Background: The chemoautotrophic bacterium Ralstonia eutropha can utilize H 2/CO 2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO 2, little work has been done to develop genetic part libraries to enable such endeavors. Results: We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters P BAD, T7, P xyls/PM, P lacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion: We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts asmore » well.« less

Authors:
 [1];  [2];  [1];  [3];  [1];  [1];  [1];  [1]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); National Taiwan Univ., Taipei (Taiwan)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1511376
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Bi, Changhao, Su, Peter, Müller, Jana, Yeh, Yi-Chun, Chhabra, Swapnil R., Beller, Harry R., Singer, Steven W., and Hillson, Nathan J. Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production. United States: N. p., 2013. Web. doi:10.1186/1475-2859-12-107.
Bi, Changhao, Su, Peter, Müller, Jana, Yeh, Yi-Chun, Chhabra, Swapnil R., Beller, Harry R., Singer, Steven W., & Hillson, Nathan J. Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production. United States. doi:10.1186/1475-2859-12-107.
Bi, Changhao, Su, Peter, Müller, Jana, Yeh, Yi-Chun, Chhabra, Swapnil R., Beller, Harry R., Singer, Steven W., and Hillson, Nathan J. Fri . "Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production". United States. doi:10.1186/1475-2859-12-107. https://www.osti.gov/servlets/purl/1511376.
@article{osti_1511376,
title = {Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production},
author = {Bi, Changhao and Su, Peter and Müller, Jana and Yeh, Yi-Chun and Chhabra, Swapnil R. and Beller, Harry R. and Singer, Steven W. and Hillson, Nathan J.},
abstractNote = {Background: The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. Results: We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion: We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.},
doi = {10.1186/1475-2859-12-107},
journal = {Microbial Cell Factories},
number = 1,
volume = 12,
place = {United States},
year = {2013},
month = {9}
}

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