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

VL - 13

SP - 194

EP - 203

JO - Metabolic Engineering

JF - Metabolic Engineering

SN - 1096-7176

IS - 2

ER -