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dc.contributor.authorCourtade, Gaston
dc.contributor.authorLe, Simone Balzer
dc.contributor.authorSætrom, Gerd Inger
dc.contributor.authorBrautaset, Trygve
dc.contributor.authorAachmann, Finn Lillelund
dc.date.accessioned2017-12-21T14:43:01Z
dc.date.available2017-12-21T14:43:01Z
dc.date.created2017-02-21T09:45:47Z
dc.date.issued2017
dc.identifier.issn0008-6215
dc.identifier.urihttp://hdl.handle.net/11250/2473600
dc.description.abstractLytic polysaccharide monooxygenases (LPMOs) are key enzymatic players of lignocellulosic biomass degradation processes. As such, they have been introduced in cellulolytic cocktails for more efficient and less expensive lignocellulose saccharification. The recombinant production of LPMOs in bacteria for scientific investigations using vectors typically based on the T7 and lacUV5 promoters has been hampered by low yields. Reasons for this have been catabolite repression when producing the proteins in defined media with glucose as the sole carbon source, as well as the lack of an inducible expression system that allows controlled production of LPMOs that are correctly processed during translocation to the periplasmic space. A cassette vector design containing the XylS/Pm system was constructed and evaluated, showing that the expression cassette could easily be used for exchanging LPMO coding genes with or without signal sequences. The cassette was shown to reliably produce mature (translocated) LPMOs under controlled conditions that were achieved by using a low dosage (0.1 mM) of the Pm inducer m-toluic acid and a low (16 °C) cultivation temperature after induction. Furthermore, the signal sequences of five bacterial LPMOs were tested, and the signal sequence of LPMO10A from Serratia marcescens was found to give highest levels of recombinant protein production and translocation. The LPMO expression cassette was also evaluated in cultivations using defined media with glucose as the sole carbon source with a product yield of 7–22 mg per L of culture in shaking flasks. The integrity of the recombinant proteins were analyzed using NMR spectroscopy, showing that the system produced correctly processed and folded LPMOs. Finally, high cell-density cultivations of the recombinant strains were carried out, demonstrating stable protein production levels at similar relative yields (42–1298 mg per L of culture; 3.8–11.6 mg per OD600nm unit) as in shaking flasks, and showing the scale-up potential of the system.nb_NO
dc.language.isoengnb_NO
dc.publisherElseviernb_NO
dc.titleA novel expression system for lytic polysaccharide monooxygenasesnb_NO
dc.typeJournal articlenb_NO
dc.typePeer reviewednb_NO
dc.description.versionpublishedVersionnb_NO
dc.source.journalCarbohydrate Researchnb_NO
dc.identifier.doi10.1016/j.carres.2017.02.003
dc.identifier.cristin1452627
dc.relation.projectNorges forskningsråd: 226244nb_NO
dc.relation.projectNorges forskningsråd: 221576nb_NO
dc.description.localcodeThis article will not be available due to copyright restrictions (c) 2017 by Elseviernb_NO
cristin.unitcode194,66,15,0
cristin.unitnameInstitutt for bioteknologi
cristin.ispublishedtrue
cristin.fulltextoriginal
cristin.qualitycode1


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