Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth

被引:69
作者
Mattozzi, Matthew D. [1 ,2 ]
Ziesack, Marika [1 ,2 ,3 ]
Voges, Mathias J. [1 ,2 ,4 ]
Silver, Pamela A. [1 ,2 ]
Way, Jeffrey C. [1 ,2 ]
机构
[1] Harvard Univ, Wyss Inst Biolog Inspired Engn, Boston, MA 02115 USA
[2] Harvard Univ, Sch Med, Dept Syst Biol, Boston, MA 02115 USA
[3] Heidelberg Univ, Inst Pharm & Mol Biotechnol, Heidelberg, Germany
[4] Delft Univ Technol, Dept Biotechnol, Delft, Netherlands
关键词
Metabolic engineering; Carbon fixation; 3-hydroxypropionate; Chloroflexus aurantiacus; E; coli; Diaminopimelic acid; Propionate biosensor; Propionate toxicity; ESCHERICHIA-COLI; PROTEIN-PRODUCTION; COA CARBOXYLASE; PROPIONYL-COA; KEY ENZYME; CYCLE; COENZYME; BIOSYNTHESIS; SYSTEM; POLY(3-HYDROXYPROPIONATE-CO-4-HYDROXYBUTYRATE);
D O I
10.1016/j.ymben.2013.01.005
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
The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetylCoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO2-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD(-) double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC(-)(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer. (C) 2013 Elsevier Inc. All rights reserved.
引用
收藏
页码:130 / 139
页数:10
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