In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli.
Biotechnol Bioeng
; 111(7): 1396-405, 2014 Jul.
Article
em En
| MEDLINE
| ID: mdl-24473754
ABSTRACT
Approaches using metabolic engineering and synthetic biology to overproduce terpenoids, such as the precursors of taxol and artemisinin, in microbial systems have achieved initial success. However, due to the lack of steady-state kinetic information and incomplete understanding of the terpenoid biosynthetic pathway, it has been difficult to build a highly efficient, universal system. Here, we reconstituted the mevalonate pathway to produce farnesene (a precursor of new jet fuel) in vitro using purified protein components. The information from this in vitro reconstituted system guided us to rationally optimize farnesene production in E. coli by quantitatively overexpressing each component. Targeted proteomic assays and intermediate assays were used to determine the metabolic status of each mutant. Through targeted engineering, farnesene production could be increased predictably step by step, up to 1.1 g/L (â¼ 2,000 fold) 96 h after induction at the shake-flask scale. The strategy developed to release the potential of the mevalonate pathway for terpenoid overproduction should also work in other multistep synthetic pathways.
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1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Escherichia coli
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Redes e Vias Metabólicas
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Ácidos Graxos Insaturados
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Engenharia Metabólica
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Ácido Mevalônico
Idioma:
En
Revista:
Biotechnol Bioeng
Ano de publicação:
2014
Tipo de documento:
Article