title: Targeted proteomics for metabolic pathway optimization: application to terpene production.
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).
authors.affiliation: Lawrence Berkeley National Laboratory, Physical Biosciences Division, Joint BioEnergy Institute, Emeryville, CA 94608, USA.
authors.forename: Alyssa M
authors.initials: AM
authors.lastname: Redding-Johanson
authors.forename: Tanveer S
authors.initials: TS
authors.lastname: Batth
authors.forename: Rossana
authors.initials: R
authors.lastname: Chan
authors.forename: Rachel
authors.initials: R
authors.lastname: Krupa
authors.forename: Heather L
authors.initials: HL
authors.lastname: Szmidt
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.forename: Taek Soon
authors.initials: TS
authors.lastname: Lee
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008798:Mevalonic Acid / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism; D000081223:Polycyclic Sesquiterpenes; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D000081223:Polycyclic Sesquiterpenes; D029701:Saccharomyces cerevisiae Proteins; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase; D008798:Mevalonic Acid