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


RESULTS
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.


CONCLUSIONS
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.
School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand. calcotmark@gmail.com.
Mark J
MJ
Calcott
School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand. david.ackerley@vuw.ac.nz.|Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand. david.ackerley@vuw.ac.nz.|Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland, New Zealand. david.ackerley@vuw.ac.nz.
David F
DF
Ackerley
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; C091136:pyoverdine synthetase protein, Pseudomonas