Abstract
BackgroundThe liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed. Solvent-producing bacteria have the capacity to convert acetate to the less toxic product acetone, but to the best of our knowledge, this trait has not been transferred to an organism that produces ethanol at high yield.ResultsWe have engineered a five-step metabolic pathway to convert acetic acid to acetone in the thermophilic anaerobe Thermoanaerobacterium saccharolyticum. The first steps of the pathway, a reversible conversion of acetate to acetyl-CoA, are catalyzed by the native T. saccharolyticum enzymes acetate kinase and phosphotransacetylase. ack and pta normally divert 30% of catabolic carbon flux to acetic acid; however, their re-introduction in evolved ethanologen strains resulted in virtually no acetic acid production. Conversion between acetic acid and acetyl-CoA remained active, as evidenced by rapid 13C label transfer from exogenous acetate to ethanol. Genomic re-sequencing of six independently evolved ethanologen strains showed convergent mutations in the hfs hydrogenase gene cluster, which when transferred to wildtype T. saccharolyticum conferred a low acid production phenotype. Thus, the mutated hfs genes effectively separate acetic acid production and consumption from central metabolism, despite their intersecting at the common intermediate acetyl-CoA. To drive acetic acid conversion to a less inhibitory product, the enzymes thiolase, acetoacetate:acetate CoA-transferase, and acetoacetate decarboxylase were assembled in T. saccharolyticum with genes from thermophilic donor organisms that do not natively produce acetone. The resultant strain converted acetic acid to acetone and ethanol while maintaining a metabolic yield of 0.50 g ethanol per gram carbohydrate.ConclusionsConversion of acetic acid to acetone results in improved ethanol productivity and titer and is an attractive low-cost solution to acetic acid inhibition.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0257-4) contains supplementary material, which is available to authorized users.
Highlights
The liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed
From an electron-based perspective, nicotinamide adenine dinucleotide (NADH) generated during glycolysis and reduced ferredoxin generated during pyruvate cleavage must be re-oxidized through reduction of acetyl-coenzyme A (CoA) to ethanol, rather than through formation of lactic acid or molecular hydrogen (Figure 1)
In attempts to engineer ethanologen strains with improved properties for industrial fermentation, we developed multiple strain lineages derived from strain M0355, a Δldh Δack Δpta mutant [16]
Summary
The liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed. Acetic acid has consistently been reported as one of the most significant inhibitors, followed by furfural, hydroxymethylfurfural, and phenolic compounds [2,3]. Unlike other inhibitors, which are by-products of specific chemical and physical pretreatment processes [4,5], acetic acid can result directly from acetylated carbohydrates, and its production is unavoidable during carbohydrate fermentation. Acetic acid is a potent inhibitor at pH values under 6, a range common for industrial fermentation [6,7,8]. The net result is both uncoupling of the transmembrane pH gradient and an accumulation of acetate anion in the cytoplasm [9,10]
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