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

被引：98

作者：

Bi, Changhao ^{[1
]}

Su, Peter ^{[1
,2
]}

Mueller, Jana ^{[1
]}

Yeh, Yi-Chun ^{[1
,3
]}

Chhabra, Swapnil R. ^{[1
]}

Beller, Harry R. ^{[4
]}

Singer, Steven W. ^{[4
]}

Hillson, Nathan J. ^{[1
]}

机构：

[1] Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Phys Biosci Div, Berkeley, CA 94720 USA

[2] Univ Calif Berkeley, Dept Chem & Biomol Engn, Berkeley, CA 94720 USA

[3] Natl Taiwan Normal Univ, Taipei, Taiwan

[4] Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Div Earth Sci, Berkeley, CA 94720 USA

来源：

MICROBIAL CELL FACTORIES | 2013年 / 12卷

关键词：

Broad-host; Synthetic biology; Ralstonia eutropha; Hydrocarbon; Chemolithoautotroph; RANGE VECTORS; GENE; EXPRESSION; H16; PROMOTERS; COMPLEX; PROTEIN; CLONING; DESIGN; SYSTEM;

D O I：

10.1186/1475-2859-12-107

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Background: The chemoautotrophic bacterium Ralstonia eutropha can utilize H-2/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 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 as well.

引用

页数：10

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