Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius
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Date
Authors
Van Zyl, Leonardo J.
Taylor, M.P.
Eley, K.
Tuffin, Marla I.
Cowan, Don A.
Journal Title
Journal ISSN
Volume Title
Publisher
Springer
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.
Description
Keywords
Pyruvate decarboxylase, Bioethanol, Gluconobacter spp., Thermophilic expression
Sustainable Development Goals
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.