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

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 10 5 M -1 s -1 at pH 5), a pH opt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C (the highest yet reported for a b a c t e r i a l P D C ) . D u e t o g o o d 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.

Genetic engineering of Geobacillus spp.

“…The expression of codon harmonized pdc in G. thermoglucosidasius (ldh − mutant) yielded 0.35 g ethanol per gram of glucose. However, ethanol production was achievable only at temperatures up to 48°C (Van Zyl et al, 2014), while the strain TM242 described earlier was able to generate ethanol even at 60°C (Cripps et al, 2009). This is a good indication that further research must be done to improve ethanol formation through PDC-dependent pathway.…”

“…Some genes derived from phylogenetically distant hosts require sequence modification to match the codon frequencies of its expression host (Blanchard et al, 2014;van Zyl et al, 2014).…”

“…The gene encoding this enzyme (pdc) is rare in bacteria and is found only in several mesophiles. The first attempts to exploit pdc for ethanol production in geobacilli did not give the expected results (Taylor et al, 2008;Thompson et al, 2008), presumably because of incompatibility of codon usage between the host and the source of the gene (Van Zyl et al, 2014). An advance was made by Van Zyl et al, who applied codon harmonization for the expression of pdc from Gluconobacter oxydans in G. thermoglucosidasius.…”

Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production

“…This design eliminated mixed‐acid fermentation and resulted in ethanol being produced at high yields. In addition, a number of studies have explored designs involving the expression of pyruvate decarboxylase from different heterologous hosts (Taylor et al, ; Thompson et al, ; Van Zyl et al, ). However, no increases in ethanol productivity were observed during growth at high (∼60°C) temperatures.…”

Recent Advances in Second Generation Ethanol Production by Thermophilic Bacteria

“…This mutant also showed good yields on xylose (1.33 mol/mol) and good productivity rates. Geobacillus thermoglucosidasius has recently be genetically modified by expressing pyruvate decarboxylase from Gluconobacter oxydans [115]. Ethanol yields obtained were as high as 1.37 mol ethanol/mol glucose.…”