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dc.contributor.authorTong, Xiaoxue
dc.contributor.authorBarberi, Tania Triscari
dc.contributor.authorBotting, Catherine H.
dc.contributor.authorSharma, Sunil V.
dc.contributor.authorSimmons, Mark J. H.
dc.contributor.authorOverton, Tim W.
dc.contributor.authorGoss, Rebecca J. M.
dc.date.accessioned2016-10-24T13:30:15Z
dc.date.available2016-10-24T13:30:15Z
dc.date.issued2016-10-21
dc.identifier.citationTong , X , Barberi , T T , Botting , C H , Sharma , S V , Simmons , M J H , Overton , T W & Goss , R J M 2016 , ' Rapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilm ' , Microbial Cell Factories , vol. 15 , no. 1 , 180 . https://doi.org/10.1186/s12934-016-0579-3en
dc.identifier.issn1475-2859
dc.identifier.otherPURE: 247036803
dc.identifier.otherPURE UUID: 24d4f362-1760-4fb6-b2e3-f6704d05a83c
dc.identifier.otherRIS: Tong2016
dc.identifier.otherScopus: 84992096639
dc.identifier.otherWOS: 000385728400003
dc.identifier.urihttp://hdl.handle.net/10023/9692
dc.descriptionThis work was supported by BBSRC Grant BB/I008713/2 and European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC Grant agreement no 614779) to Goss and BB/I006834/1 to Simmons.en
dc.description.abstractBackground: Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm’s extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme’s longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. Results: Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. Conclusions: Here we provide the first evidence of a recombinant enzyme’s regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a “plug-and-play” fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals.
dc.format.extent11
dc.language.isoeng
dc.relation.ispartofMicrobial Cell Factoriesen
dc.rightsThe Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.en
dc.subjectEngineered E, coli biofilmen
dc.subjectBiotransformationen
dc.subjectBiocatalysisen
dc.subjectTryptophan synthaseen
dc.subjectHalotryptophanen
dc.subjectQD Chemistryen
dc.subjectNDASen
dc.subject.lccQDen
dc.titleRapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilmen
dc.typeJournal articleen
dc.description.versionPublisher PDFen
dc.contributor.institutionUniversity of St Andrews.School of Chemistryen
dc.contributor.institutionUniversity of St Andrews.Biomedical Sciences Research Complexen
dc.contributor.institutionUniversity of St Andrews.EaSTCHEMen
dc.identifier.doihttps://doi.org/10.1186/s12934-016-0579-3
dc.description.statusPeer revieweden


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