Targeted proteomics for metabolic pathway optimization: application to terpene production

Research output: Contribution to journalJournal articleResearchpeer-review

  • Alyssa M Redding-Johanson
  • Batth, Tanveer Singh
  • Rossana Chan
  • Rachel Krupa
  • Heather L Szmidt
  • Paul D Adams
  • Jay D Keasling
  • Taek Soon Lee
  • Aindrila Mukhopadhyay
  • Christopher J Petzold

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).

Original languageEnglish
JournalMetabolic Engineering
Issue number2
Pages (from-to)194-203
Number of pages10
Publication statusPublished - Mar 2011

    Research areas

  • Escherichia coli/genetics, Escherichia coli Proteins/genetics, Fermentation/genetics, Gene Expression Regulation, Bacterial, Genetic Engineering, Metabolic Networks and Pathways/genetics, Mevalonic Acid/metabolism, Phosphotransferases (Alcohol Group Acceptor)/metabolism, Phosphotransferases (Phosphate Group Acceptor)/metabolism, Proteomics/methods, Saccharomyces cerevisiae/genetics, Saccharomyces cerevisiae Proteins/genetics, Sesquiterpenes/metabolism

ID: 204046966