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

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dc.contributor.author Van Zyl, Leonardo J.
dc.contributor.author Taylor, M.P.
dc.contributor.author Eley, K.
dc.contributor.author Tuffin, Marla I.
dc.contributor.author Cowan, Don A.
dc.date.accessioned 2014-07-04T10:35:30Z
dc.date.issued 2014-02
dc.description.abstract This study reports the expression, purification and kinetic characterization of a PDC from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120μM at pH 5), high catalytic efficiency (4.75 x 105 M-1s-1 at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C (the highest yet reported for a bacterial PDC). Due to good in vitro thermostablity (approximately 40% enzyme activity retained after 30 minutes at 65°C) this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius. Initial studies using a variety of methods failed to detect activity at any growth temperature. 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 successful expression of PDC in a true thermophile. Yields as high as 0.35 g/g ±0.04 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 that the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription / translation coupling. en_US
dc.description.librarian hb2014 en_US
dc.description.sponsorship National Research Foundation South Africa en_US
dc.description.uri http://link.springer.com/journal/253 en_US
dc.identifier.citation Van Zyl, LJ, Taylor, MP, Eley, K., Tuffin, M & Cowan, DA 2014, 'Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius', Applied Microbiology and Biotechnology, vol. 98, no. 3, pp. 1247-1259. en_US
dc.identifier.issn 0175-7598 (print)
dc.identifier.issn 1432-0614 (online)
dc.identifier.other 10.1007/s00253-013-5380-1
dc.identifier.uri http://hdl.handle.net/2263/40553
dc.language.iso en en_US
dc.publisher Springer en_US
dc.rights © Springer-Verlag Berlin Heidelberg 2013. The original publication is available at : http://link.springer.com/journal/253 en_US
dc.subject Pyruvate decarboxylase en_US
dc.subject Bioethanol en_US
dc.subject Gluconobacter spp. en_US
dc.subject Thermophilic expression en_US
dc.title Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius en_US
dc.type Postprint Article en_US


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