Targeted proteomics for metabolic pathway optimization: application to terpene production.

Autor: Redding-Johanson AM; Lawrence Berkeley National Laboratory, Physical Biosciences Division, Joint BioEnergy Institute, Emeryville, CA 94608, USA., Batth TS, Chan R, Krupa R, Szmidt HL, Adams PD, Keasling JD, Lee TS, Mukhopadhyay A, Petzold CJ
Jazyk: angličtina
Zdroj: Metabolic engineering [Metab Eng] 2011 Mar; Vol. 13 (2), pp. 194-203. Date of Electronic Publication: 2011 Jan 05.
DOI: 10.1016/j.ymben.2010.12.005
Abstrakt: 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).
(Copyright © 2011 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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