Thermoanaerobacterium saccharolyticum JW/SL-YS485 is a gram positive, thermophilic, obligate anaerobe that grows in a temperature range of 37°C - 66°C and a pH range of 3.9 – 6.5. This organism can consume the majority of hydrolysis sugars found in lignocellulosic biomass as well as xylan and starch, but not cellulose. It produces ethanol, acetic acid, lactic acid, hydrogen, and carbon dioxide as fermentation products.

By using homology based gene knockout to target the acetate kinase (ack) and L-lactate dehydrogenase (L-ldh) genes, the strain ALK2 was created that produces ethanol via a high yield pathway featuring the enzymes pyruvate:ferredoxin oxidoreductase, ferredoxin:NAD oxidoreductase, acetaldehyde dehydrogenase, and alcohol dehydrogenase.  T. saccharolyticum strain ALK2 can be used in a thermophilic simultaneous saccharification and fermentation (tSSF) process that can reduce cellulase requirements as a result of operating at the optimal conditions for enzymatic hydrolysis.  We show that utilization of this organism at 50°C lowers the requirement for added cellulase 2.5-fold relative to SSF at 37°C with S. cerevisiae.

In addition to curtailing acetic acid production by deletion of ack, an alternate engineering strategy is presented wherein acetic acid metabolic flux is re-directed to lactic acid and ethanol by deletion of the hydrogenase enzyme.  By removing the organism’s ability to produce hydrogen, it is obligated cease acetic acid production in order to balance electron flux.  A strain carrying deletions of the hydrogenase and L-ldh genes produces ethanol at a similarly high yield as the ack and L-ldh minus strain.