Heterologous expression of pyruvate decarboxylase in Geobacillus thermoglucosidasius

Abstract

Expression of a pyruvate decarboxylase (Pdc) pathway in metabolically versatile thermophilic bacteria could create novel ethanologenic organisms, but no suitable thermostable Pdc is available. We have demonstrated that Pdc from Zymomonas mobilis can be expressed in an active form in Geobacillus thermoglucosidasius at up to 52 degrees C, while expression of Pdc polypeptides up to 54 degrees C was evident from Western blotting. By using an unstable lactate dehydrogenase (ldh) mutant of G. thermoglucosidasius, indirect evidence of Pdc activity in vivo was also obtained.

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

“…Production of soluble ZmoPDC in cell free extracts of G. thermoglucosidasius grown at 52°C, 54°C, 56°C and 58°C was observed to decrease with increased temperature and PDC activity was undetectable above 52°C (Thompson et al, 2008). For ZpaPDC, activity was absent at growth temperatures above 45°C (Taylor et al, 2008), while in this study, the GoxPDC WT showed no activity at 45°C (see above).…”

“…When correctly folded the GoxPDC protein displays relatively high thermostability when assayed in vitro. However, this does not necessarily translate to the ability to fold correctly at elevated temperatures, offering a possible explanation for the apparent failure of functional expression at 52°C, unlike ZmoPDC when expressed in the same host (Thompson et al, 2008). We suggest that although codon optimization contributes to the correct folding of a nascent protein during translation of the mRNA, it cannot necessarily compensate for the kinetics involved in protein folding in temperature ranges outside those for which the protein had been selected for or evolved under.…”

“…The reasons for the low levels of activity are not fully understood (Taylor et al, 2008;Thompson et al, 2008), but protein misfolding resulting in inactive protein has been proposed (Thompson et al ., 2008). Attempts to express these proteins in mesophilic Gram positive hosts (notably lactic acid bacteria and Bacillus megatarium) have also had limited success (Gold et al, 1996;Bongers et al, 2005;Kaczowka et al, 2005;Liu et al, 2005;Talarico et al, 2005;Liu et al, 2006;Liu et al, 2007;Orencio-Trejo et al, 2008;Bi et al, 2009).…”

Extremophiles and their Application to Biofuel Research

“…Strategies involved the metabolic engineering of endogenous pathways to improve ethanol yield (Cripps et al, 2009) and the de novo expression of pyruvate decarboxylases (pdc) from mesophilic gram-negative species such as Zymomonas mobilis (Taylor et al, 2008;Thompson et al, 2008). The latter strategy has been used with great success in Escherichia coli and other enterobacteria (Ohta et al, 1991a,b).…”

“…The latter strategy has been used with great success in Escherichia coli and other enterobacteria (Ohta et al, 1991a,b). In G. thermoglucosidasius, however, although the Z. mobilispdc was expressed successfully, protein misfolding hindered pdc expression at temperatures close to the T opt (66 • C) value of the strain (Thompson et al, 2008). More recently, studies have focused on the development of genetic methods for the evolution of ethanol-producing variants (Cripps et al, 2009) led by an industrial company, TMO Renewables Ltd. (http://www.tmo-group.com).…”