title: Application of targeted proteomics to metabolically engineered Escherichia coli.
abstract: As synthetic biology matures to compete with chemical transformation of commodity and high-value compounds, a wide variety of well-characterized biological parts are needed to facilitate system design. Protein quantification based on selected-reaction monitoring (SRM) mass spectrometry compliments metabolite and transcript analysis for system characterization and optimizing flux through engineered pathways. By using SRM quantification, we assayed red fluorescent protein (RFP) expressed from plasmids containing several inducible and constitutive promoters and subsequently assessed protein production from the same promoters driving expression of eight mevalonate pathway proteins in Escherichia coli. For each of the promoter systems, the protein level for the first gene in the operon followed that of RFP, however, the levels of proteins produced from genes farther from the promoter were much less consistent. Second, we used targeted proteomics to characterize tyrosine biosynthesis pathway proteins after removal of native regulation. The changes were not expected to cause significant impact on protein levels, yet significant variation in protein abundance was observed and tyrosine production for these strains spanned a range from less than 1 mg/L to greater than 250 mg/L. Overall, our results underscore the importance of targeted proteomics for determining accurate protein levels in engineered systems and fine-tuning metabolic pathways.
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA.
authors.forename: Pragya
authors.initials: P
authors.lastname: Singh
authors.forename: Tanveer S
authors.initials: TS
authors.lastname: Batth
authors.forename: Darmawi
authors.initials: D
authors.lastname: Juminaga
authors.forename: Robert H
authors.initials: RH
authors.lastname: Dahl
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D014644:Genetic Variation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D060847:Metabolic Engineering / Q000379:methods*; D008798:Mevalonic Acid / Q000378:metabolism; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D014169:Transformation, Bacterial; D014443:Tyrosine / Q000096:biosynthesis / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D008164:Luminescent Proteins; D008798:Mevalonic Acid; D014443:Tyrosine; D000097573:Red Fluorescent Protein