Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

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dc.contributor.authorVan Zyl, L.J.
dc.contributor.authorTaylor, M.P.
dc.contributor.authorEley, K.
dc.contributor.authorTuffin, Marla
dc.contributor.authorCowan, Donald A.
dc.date.accessioned2018-03-01T12:26:21Z
dc.date.available2018-03-01T12:26:21Z
dc.date.issued2013
dc.description.abstractThis study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans . Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75×105 M−1 s−1 at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45–55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host’s transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35±0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.en_US
dc.description.accreditationWeb of Science
dc.identifier.citationVan Zyl, L.J. et al. (2013). Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius. Applied Microbiology Biotechnology, 98: 1247 – 1259en_US
dc.identifier.issn0175-7598
dc.identifier.urihttp://dx.doi.org/10.1007/s00253-013-5380-1
dc.identifier.urihttp://hdl.handle.net/10566/3557
dc.language.isoenen_US
dc.privacy.showsubmitterFALSE
dc.publisherSpringer Verlagen_US
dc.status.ispeerreviewedTRUE
dc.status.ispeerreviewedThis is the author-version of the article published online at: http://dx.doi.org/10.1007/s00253-013-5380-1
dc.subjectPyruvate decarboxylaseen_US
dc.subjectBioethanolen_US
dc.subjectGluconobacter spp.en_US
dc.subjectThermophilic expressionen_US
dc.titleEngineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasiusen_US
dc.typeArticleen_US

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