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

Microbial Cell Factories

Research

Harry R Beller¹  Steven W Singer¹  Jana Müller²  Swapnil R Chhabra²  Nathan J Hillson²  Peter Su³  Yi-Chun Yeh⁴  Changhao Bi⁵ 

[1] Earth Sciences Division, Lawrence Berkeley National Laboratory, 94720, Berkeley, CA, USA;Physical Biosciences Division, Lawrence Berkeley National Laboratory, 94720, Berkeley, CA, USA;Physical Biosciences Division, Lawrence Berkeley National Laboratory, 94720, Berkeley, CA, USA;Department of Chemical & Biomolecular Engineering, University of California, 94720, Berkeley, CA, USA;Physical Biosciences Division, Lawrence Berkeley National Laboratory, 94720, Berkeley, CA, USA;National Taiwan Normal University, Taipei, Taiwan;Physical Biosciences Division, Lawrence Berkeley National Laboratory, 94720, Berkeley, CA, USA;Tianjin Institute of Biotechnology, Chinese Academy of Sciences, Tianjin, China;

关键词: Broad-host;    Synthetic biology;    Ralstonia eutropha;    Hydrocarbon;    Chemolithoautotroph;   

DOI  :  10.1186/1475-2859-12-107

 received in 2013-09-13, accepted in 2013-11-11,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsWe 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.ConclusionWe constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.

【 授权许可】

Unknown   
© Bi et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.