Anaerobic detoxification of acetic acid in a thermophilic ethanologen

作者：Shaw A Joe; Miller Bethany B; Rogers Stephen R; Kenealy William R; Meola Alex; Bhandiwad Ashwini; Sillers W Ryan; Shikhare Indraneel; Hogsett David A; Herring Christopher D^*

来源：Biotechnology for Biofuels, 2015, 8(1): 75.

DOI：10.1186/s13068-015-0257-4

• Acetate detoxification
• Cellulosic ethanol
• Metabolic engineering
• Inhibitors

Background: The 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.
Results: We 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 Thermoanaerobacterium 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 C-13 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 Thermoanaerobacterium 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 Thermoanaerobacterium 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.
Conclusions: Conversion of acetic acid to acetone results in improved ethanol productivity and titer and is an attractive low-cost solution to acetic acid inhibition.

• 出版日期2015-5-9