Targeted proteomics for metabolic pathway optimization: application to terpene production.
Metab Eng
; 13(2): 194-203, 2011 Mar.
Article
em En
| MEDLINE
| ID: mdl-21215324
ABSTRACT
Successful metabolic engineering relies on methodologies that aid assembly and optimization of novel pathways in microbes. Many different factors may contribute to pathway performance, and problems due to mRNA abundance, protein abundance, or enzymatic activity may not be evident by monitoring product titers. To this end, synthetic biologists and metabolic engineers utilize a variety of analytical methods to identify the parts of the pathway that limit production. In this study, targeted proteomics, via selected-reaction monitoring (SRM) mass spectrometry, was used to measure protein levels in Escherichia coli strains engineered to produce the sesquiterpene, amorpha-4,11-diene. From this analysis, two mevalonate pathway proteins, mevalonate kinase (MK) and phosphomevalonate kinase (PMK) from Saccharomyces cerevisiae, were identified as potential bottlenecks. Codon-optimization of the genes encoding MK and PMK and expression from a stronger promoter led to significantly improved MK and PMK protein levels and over three-fold improved final amorpha-4,11-diene titer (>500 mg/L).
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sesquiterpenos
/
Proteínas de Escherichia coli
/
Proteômica
/
Escherichia coli
/
Redes e Vias Metabólicas
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Metab Eng
Assunto da revista:
ENGENHARIA BIOMEDICA
/
METABOLISMO
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos