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Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases

Overview of attention for article published in BMC Microbiology, August 2015
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Title
Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases
Published in
BMC Microbiology, August 2015
DOI 10.1186/s12866-015-0496-3
Pubmed ID
Authors

Mark J. Calcott, David F. Ackerley

Abstract

Non-ribosomal peptide synthetase (NRPS) enzymes govern the assembly of amino acids and related monomers into peptide-like natural products. A key goal of the field is to develop methods to effective recombine NRPS domains or modules, and thereby generate modified or entirely novel products. We previously showed that substitution of the condensation (C) and adenylation (A) domains in module 2 of the pyoverdine synthetase PvdD from Pseudomonas aeruginosa led to synthesis of modified pyoverdines in a minority of cases, but that more often the recombinant enzymes were non-functional. One possible explanation was that the majority of introduced C domains were unable to effectively communicate with the thiolation (T) domain immediately upstream, in the first module of PvdD. To test this we first compared the effectiveness of C-A domain substitution relative to T-C-A domain substitution using three different paired sets of domains. Having previously demonstrated that the PvdD A/T domain interfaces are tolerant of domain substitution, we hypothesised that T-C-A domain substitution would lead to more functional recombinant enzymes, by maintaining native T/C domain interactions. Although we successfully generated two recombinant pyoverdines, having a serine or a N5-formyl-N5-hydroxyornithine residue in place of the terminal threonine of wild type pyoverdine, in neither case did the T-C-A domain substitution strategy lead to substantially higher product yield. To more comprehensively examine the abilities of non-native T domains to communicate effectively with the C domain of PvdD module 2 we then substituted the module 1 T domain with 18 different T domains sourced from other pyoverdine NRPS enzymes. In 15/18 cases the recombinant NRPS was functional, including 6/6 cases where the introduced T domain was located upstream of a C domain in its native context. Our data indicate that T domains are generally able to interact effectively with non-native C domains, contrasting with previous findings that they are not generally portable upstream of epimerisation (E) or thioesterase (TE) domains. This offers promise for NRPS recombination efforts, but also raises the possibility that some C domains are unable to efficiently accept non-native peptides at their donor site due to steric constraints or other limitations.

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The data shown below were compiled from readership statistics for 40 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Japan 1 3%
Unknown 39 98%

Demographic breakdown

Readers by professional status Count As %
Student > Bachelor 9 23%
Student > Master 8 20%
Student > Ph. D. Student 5 13%
Researcher 4 10%
Lecturer 2 5%
Other 2 5%
Unknown 10 25%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 14 35%
Agricultural and Biological Sciences 7 18%
Chemistry 3 8%
Computer Science 1 3%
Neuroscience 1 3%
Other 1 3%
Unknown 13 33%
Attention Score in Context

Attention Score in Context

This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 14 August 2015.
All research outputs
#14,234,315
of 22,821,814 outputs
Outputs from BMC Microbiology
#1,449
of 3,190 outputs
Outputs of similar age
#136,414
of 264,395 outputs
Outputs of similar age from BMC Microbiology
#20
of 48 outputs
Altmetric has tracked 22,821,814 research outputs across all sources so far. This one is in the 35th percentile – i.e., 35% of other outputs scored the same or lower than it.
So far Altmetric has tracked 3,190 research outputs from this source. They receive a mean Attention Score of 4.1. This one has gotten more attention than average, scoring higher than 50% of its peers.
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We're also able to compare this research output to 48 others from the same source and published within six weeks on either side of this one. This one is in the 45th percentile – i.e., 45% of its contemporaries scored the same or lower than it.