title: Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius.
abstract: This 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 × 10(5) 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.
authors.affiliation: Institute for Microbial Biotechnology and Metagenomics (IMBM), University of the Western Cape, Modderdam Road, Bellville, Cape Town, South Africa.
authors.forename: L J
authors.initials: LJ
authors.lastname: Van Zyl
authors.forename: M P
authors.initials: MP
authors.lastname: Taylor
authors.forename: K
authors.initials: K
authors.lastname: Eley
authors.forename: M
authors.initials: M
authors.lastname: Tuffin
authors.forename: D A
authors.initials: DA
authors.lastname: Cowan
mesh_terms: D003062:Codon; D004795:Enzyme Stability; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D056506:Geobacillus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D020600:Gluconobacter oxydans / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D060847:Metabolic Engineering / Q000379:methods*; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D013696:Temperature
chemical_list: D003062:Codon; D000431:Ethanol; D019289:Pyruvic Acid; D011767:Pyruvate Decarboxylase; D005947:Glucose