Mutant Variants of the Substrate-Binding Protein DppA from Escherichia coli Enhance Growth on Nonstandard γ-Glutamyl Amide-Containing Peptides

Abstract

The import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) of is an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA. characterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides. Fundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.

Defining the Environmental Adaptations of Genus Devosia: Insights into its Expansive Short Peptide Transport System and Positively Selected Genes

“…A relatively high diversity of the substrate binding proteins in Devosia unveiled the high nutritional demands and efficiency of the genus towards uptake of a wide range of structurally and chemically diverse amino acid side chains from environment. Apart from nutritional significance, the permeases are also gates to acquire natural and non-natural cargo molecules attached with amino acid side chains of peptides thereby acting as environmental sensors 37,38 . These signals drive the bacterial chemotaxis and form the basis of bacterial tolerance and bioremediation of environmental pollutants by bacteria 39 .…”

Comprehensive elucidation of glutathione import inEscherichia coli

“…Using GsiB as a template, a BLAST search against the E. coli proteome identified several candidates for periplasmic binding proteins of ABC transporters. Among them were the periplasmic binding proteins of more or less well-characterized ABC transporters, such as DppA with 49% similarity, known to import di-and tripeptides (Kuenzl et al, 2018;Smith et al, 1999), SapA with 43% similarity, known for the transport of antimicrobial peptides in Salmonella typhimurium (Parra-Lopez et al, 1993) and Haemophilus influenza (Shelton et al, 2011) and the export of putrescine in E. coli (Sugiyama et al, 2016), OppA (43% similarity), part of an oligopeptide transporter (Klepsch et al, 2011), and YejA (36% similarity), a putative oligopeptide transporter, which is known to confer resistance to the peptide antibiotic microcin C (Novikova et al, 2007;Vondenhoff et al, 2011) (see Figure 4A for a schematic overview). In order to test, if one of these ABC transporters is needed to import reduced glutathione, we deleted genes coding for one of the permease domains (OppC, DppC and SapC) or the periplasmic binding protein in case of Yej (YejA) in cells lacking glutathione biosynthesis and the GsiC permease.…”