Construction and expression of an ethanol production operon in Gram-positive bacteria

Abstract

Pyruvate decarboxylase (PDC), an enzyme central to homoethanol fermentation, catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon dioxide. PDC enzymes from diverse organisms have different kinetic properties, thermal stability and codon usage that are likely to offer unique advantages for the development of desirable Gram-positive biocatalysts for use in the ethanol industry. To examine this further, pdc genes from bacteria to yeast were expressed in the Gram-positive host Bacillus megaterium. The PDC activity and protein levels were determined for each strain. In addition, the levels of pdc-specific mRNA transcripts and stability of recombinant proteins were assessed. From this analysis, the pdc gene of Gram-positive Sarcina ventriculi was found to be the most advantageous for engineering high-level synthesis of PDC in a Gram-positive host. This gene was thus selected for transcriptional coupling to the alcohol dehydrogenase gene (adh) of Geobacillus stearothermophilus. The resulting Gram-positive ethanol production operon was expressed at high levels in B. megaterium. Extracts from this recombinant were shown to catalyse the production of ethanol from pyruvate.

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

“…A similar prediction that Gram negative PDCs should express well in an E. coli background and poorly in a Gram positive host is supported by the wealth of expression data of these genes in various E. coli hosts Chandra Raj et al, 2001;Talarico et al, 2001;Raj et al, 2002;Talarico et al, 2005).…”

“…No previous studies reporting the functional expression of the pdc gene alone in E. coli have shown any evidence of elevated ethanol yields (Talarico et al, 2001;Liu et al, 2005;Talarico et al, 2005).…”

“…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). …”

“…Incompatibilities in codon usage and their effect on expression of PDCs have been reported (Lowe et al, 1992;Talarico et al, 2001;Talarico et al, 2005). Two examples of the effect of codon usage on PDC production include the 5-10 fold increase in soluble SvePDC when expressed in an E. coli strain with or without accessory tRNAs (specifically those which are rarely used in E. coli), as well as the superior production of this PDC relative to those from A. pasteurianus and Z. mobilis in B. megatarium (Talarico et al, 2001;Raj et al, 2002;Talarico et al, 2005).…”

Pilot-Scale Production of Fatty Acid Ethyl Esters by an Engineered Escherichia coli Strain Harboring the p(Microdiesel) Plasmid

“…Bioethanol is of great importance in that it is itself a biofuel and is required for the transesterification process in microdiesel biosynthesis; many studies have reported on the optimization of its production (11,41,49). However, it has been also reported that ethanol causes multiple effects on E. coli K-12 cells, including reduction of growth rates and inhibition of cell division at concentrations higher than 0.6 M and loss of viability at a concentration of 1.6 M (12) and damage to the cell membrane and degradation of ribonucleic acids (36).…”