Transport and Hydrolysis of Peptides by Microorganisms

Payne, John W.

doi:10.1002/9780470720318.ch17

Cited by 17 publications

(9 citation statements)

References 49 publications

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“…Thus, the design of suitable biosensors would provide a convenient tool for strain analysis and screening, and might be especially useful for the identification of non-intuitive targets for strain improvement (Binder et al 2012;Mahr et al 2015;Mustafi et al 2012). However, our study revealed that several parameters require careful consideration to set up a screening procedure for effector-responsive promoters, including the adaption to growth medium, the uptake and catabolism of effector molecules (Luo et al 2014;Payne 1977), the activation of the gene expression machinery (Bintu et al 2005b;Carey et al 2013;Fritz et al 2014;Mäkelä et al 2013), the maturation of fluorescent proteins (Craggs 2009;Iizuka et al 2011), and last but not least, the composition of the particular growth medium. Here, we used M9 minimal medium due to its lack of other amino acids and carbon source and its very low auto-fluorescence (Miller 1972).…”

Section: Discussionmentioning

confidence: 98%

“…For counter-selection, the screening medium was prepared without Ala-Phe. We used dipeptides for screening, as previous characterizations of biosensors featured an improved uptake of short peptides-the intracellular hydrolysis of which leads to an increase of the respective effector amino acids in the cytoplasm Payne 1977;Simmonds and Griffith 1961). Within five steps of toggled selection, no significant enrichment of clones falling within the respective gate was observed.…”

Section: Screening For L-phenylalanine-responsive Promotersmentioning

confidence: 99%

“…A competitive dipeptide feeding strategy was used to describe the relationship between effector input concentrations and the biosensor output . More precisely, different ratios of effector containing dipeptides (Ala-Phe) and non-effector dipeptides (Ala-Ala) competitive for dipeptide uptake systems were added to adjust the intracellular concentration of effector molecules (Payne 1977;Vrljic et al 1996).…”

Section: Quantitative Characterization Of Different Biosensor Designsmentioning

confidence: 99%

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Screening of an Escherichia coli promoter library for a phenylalanine biosensor

Mahr

Boeselager

Wiechert

et al. 2016

Appl Microbiol Biotechnol

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In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors.

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Section: Discussionmentioning

confidence: 98%

Section: Screening For L-phenylalanine-responsive Promotersmentioning

confidence: 99%

Section: Quantitative Characterization Of Different Biosensor Designsmentioning

confidence: 99%

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Screening of an Escherichia coli promoter library for a phenylalanine biosensor

Mahr

Boeselager

Wiechert

et al. 2016

Appl Microbiol Biotechnol

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“…The E. coli peptide transporters dipeptide permease (DppABCDF) and oligopeptide permease (OppABCDF) are the main uptake routes for peptides from the environment and are known to have rather relaxed substrate specificities (13). Dipeptide permease has a preference for dipeptides and only little affinity for certain tripeptides (14,15).…”

mentioning

confidence: 99%

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

Kuenzl

Li-Blatter

Srivastava

et al. 2018

Appl Environ Microbiol

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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.

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“…Briefly, the stability constants of the complexes should be similar to some biological ligands to ensure the metal ion can be associated in a complex 50 . The complexes should also have low molecular weight (<1000 Da) to promote diffusion across biological membranes and absorption 51 . After absorption the complex must hydrolyze to release the metal ion 50,52 .…”

mentioning

confidence: 99%

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2016

IJPSR

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Amino acid chelated complexes were synthesized and characterized to determine their purity. The purity of the complexes was found to be ranging from 42-75%. The HPLC method for determination of free amino acid content in the complexes was developed and validated and found to be linear, precise and accurate within concentration range of 0.157 mg/mL to 20.14 mg/mL. The method was used to estimate the purity of the amino acid complexes. The changes in the carboxylate and amine group stretches in FT-IR indicated the association of metal ion with the amino acid. The initial studies described above demonstrated that the chelated complexes can produce sufficient osmotic pressure to be used as ergogenic aids.

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Transport and Hydrolysis of Peptides by Microorganisms

Cited by 17 publications

References 49 publications

Screening of an Escherichia coli promoter library for a phenylalanine biosensor

Screening of an Escherichia coli promoter library for a phenylalanine biosensor

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

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