Anaerobic detoxification of acetic acid in a thermophilic ethanologen.
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 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 (13)C 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.


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.
Mascoma Corporation, Lebanon, NH 03766 USA ; Novogy Inc., 85 Bolton St, Cambridge, MA 02140 USA.
A Joe
AJ
Shaw
Mascoma Corporation, Lebanon, NH 03766 USA.
Bethany B
BB
Miller
Mascoma Corporation, Lebanon, NH 03766 USA.
Stephen R
SR
Rogers
Mascoma Corporation, Lebanon, NH 03766 USA ; Verdezyne Inc., 2715 Loker Avenue West, Carlsbad, CA 92010 USA.
William R
WR
Kenealy
Mascoma Corporation, Lebanon, NH 03766 USA.
Alex
A
Meola
Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA ; Energy Biosciences Institute, 2151 Berkeley Way, Berkeley, CA 94704 USA.
Ashwini
A
Bhandiwad
Mascoma Corporation, Lebanon, NH 03766 USA ; Myriant Corporation, 66 Cummings Park, Woburn, MA 01801 USA.
W Ryan
WR
Sillers
Mascoma Corporation, Lebanon, NH 03766 USA.
Indraneel
I
Shikhare
Mascoma Corporation, Lebanon, NH 03766 USA ; OPX Biotechnologies Inc., 2425 55th Street, Boulder, CO 80301 USA.
David A
DA
Hogsett
Mascoma Corporation, Lebanon, NH 03766 USA ; Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA.
Christopher D
CD
Herring
Acetate detoxification; Cellulosic ethanol; Inhibitors; Metabolic engineering