Exploiting the Anaerobic Expression of Pyruvate Dehydrogenase for the Production of Biofuels

Abstract

The Pyruvate dehydrogense complex (PDH) is a primarily aerobic enzyme which catalyses pyruvate to acetyl-CoA and carbon dioxide. Its counterpart in anaerobic metabolism is pyruvate formate lyase (Pfl) which converts pyruvate to acetyl-CoA and formate. A novel fermentation pathway involving PDH rather than Pfl (or equivalent), which retains the reducing equivalents from pyruvate oxidation, could provide a novel route for ethanol production, as well as changes in redox balance opening up opportunities for the production of higher alcohols such as butanol. Utilising PDH for the production of biofuels has been investigated in three microorganisms: Geobacillus thermodenitrificans, Bacillus subtilis, and E. coli. Geobacillus thermodenitrificans does express Pfl, thus PDH is always active in the G. thermodenitrificans regardless of whether the bacterium is growing in aerobic or anaerobic conditions. To utilise this PDH in the production of ethanol a bi-functional alcohol dehydrogenase (AdhE) was introduced to G. thermodenitrificans K1041. Further optimisation of ethanol production was achieved by knocking-out lactate dehydrogenase (Ldh), which would otherwise compete with ethanol for flux from acetyl-CoA, and activity of the PDH promoter verses potential alternative promoters to increase the expression of the native PDH was investigated. Like G. thermodenitrificans, Bacillus subtilis also does not have a PFL pathway, but does have a native Adh so can undergo fermentation, albeit poorly. To increase ethanol production competing fermentation pathways were knocked-out, however this resulted in strains which were unable to grow anaerobically. The activity of the native PDH promoter was investigated, and PDH subsequently upregulated. The production of 1-butanol from B. subtilis was also achieved using expression of Clostridial genes encoding a butanol synthetic pathway from a plasmid and from chromosomal integrations. PDH in Gram-negative bacteria such as E. coli are not active during anerobic growth due to fermentation resulting in elevated levels of intracellular NADH; which in turn triggers negative feedback inhibition of PDH. A consequence of this is E. coli strains which are engineered to produce increased titres of ethanol by knocking-out pfl are unable to grow anaerobically. To alleviate this problem a PDH from gram-positive bacteria was expressed in E. coli. The effect of these PDH was also used to assess their potential benefits on 1-butanol in E. coli, by introducing Clostridial genes encoding a butanol synthetic pathway via plasmids.