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.

Advances in proteomics for production strain analysis

“…SRM-MS has been validated against analysis of red fluorescent protein expression levels in an expression plasmid and output of the tyrosine production pathway, controlled with a variety of different strength constitutive promoters [60]. These methods can also be coupled with quantification methods that incorporate a standard, such as QconCAT, to generate absolute protein quantification levels [15 ].…”

Engineering

“…A related work characterized the impact on SHK pathway enzyme levels resulting from the removal of TyrR regulator, along with the use of a feedback-resistant TyrA and deletion of the pheA gene on L-TYR producing strains. The results showed that small changes in protein levels caused by the genetic alterations can have a big impact on metabolite production, as a 250-fold span of L-TYR concentrations were detected [118]. A different work found many proteins differentially expressed as a response to the sole inactivation of the pykF gene, including DAHP synthase (AroG), SHK dehydrogenase (AroE), SHK kinase I (AroK), CHA synthase (AroC), prephenate dehydratase (PheA), anthranilate synthase (TrpD, TrpE) and L-TRP synthase (TrpA), as compared to the wild type strain [119].…”

Bridging the gap between systems biology and synthetic biology

“…The ease with which these assays can now be generated will enable a wide variety of new applications, potentially including whole-proteome analysis. For example, Singh et al (2012) used SRM techniques to optimize flux within the mevalonate pathway in E. coli . Malmstroem et al (2009) applied this technique to determine the average quantity of proteins per cell for 51% of the open reading frames (ORFs), or 83% of the proteome of Leptospira interrogans , a human pathogen.…”