TY - JOUR
T1 - Targeted proteomics for metabolic pathway optimization
T2 - application to terpene production
AU - Redding-Johanson, Alyssa M
AU - Batth, Tanveer S
AU - Chan, Rossana
AU - Krupa, Rachel
AU - Szmidt, Heather L
AU - Adams, Paul D
AU - Keasling, Jay D
AU - Lee, Taek Soon
AU - Mukhopadhyay, Aindrila
AU - Petzold, Christopher J
N1 - Copyright © 2011 Elsevier Inc. All rights reserved.
PY - 2011/3
Y1 - 2011/3
N2 - 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).
AB - 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).
KW - Escherichia coli/genetics
KW - Escherichia coli Proteins/genetics
KW - Fermentation/genetics
KW - Gene Expression Regulation, Bacterial
KW - Genetic Engineering
KW - Metabolic Networks and Pathways/genetics
KW - Mevalonic Acid/metabolism
KW - Phosphotransferases (Alcohol Group Acceptor)/metabolism
KW - Phosphotransferases (Phosphate Group Acceptor)/metabolism
KW - Proteomics/methods
KW - Saccharomyces cerevisiae/genetics
KW - Saccharomyces cerevisiae Proteins/genetics
KW - Sesquiterpenes/metabolism
U2 - 10.1016/j.ymben.2010.12.005
DO - 10.1016/j.ymben.2010.12.005
M3 - Journal article
C2 - 21215324
SN - 1096-7176
VL - 13
SP - 194
EP - 203
JO - Metabolic Engineering
JF - Metabolic Engineering
IS - 2
ER -