Understanding the role of the hfs hydrogenase in Thermoanaerobacterium saccharolyticum

The primary hydrogenase in T. saccharolyticum is called hfs, and it consists of 4 subunits, hfsA, hfsB, hfsC and hfsD.  Previously it has been shown that a complete deletion of all 4 subunits (A, B, C and D) does not increase ethanol production. Deletion of hfsABCD and ldh in combination increased ethanol yield by 44%.  In strains engineered for high ethanol production, spontaneous mutations are often found in one or more of the hfs subunits.  A mutant hfs operon (called hfs*), containing two point mutations from a high-ethanol-producing strain, was re-introduced into the wild-type strain resulting in a 36% increase in ethanol yield.  To better understand the hydrogenase, we deleted each subunit individually.  Deletion of hfsA or hfsB in the wild-type strain increased ethanol yield to ≥89% of the theoretical maximum.  Even in the presence of high cellobiose concentrations (20 g/l ), lactate production was not increased. Also, H₂ production was significantly decreased in DhfsA (70%) and in DhfsB (90%), which is highly correlated with acetate production. However, deletion of hfsC and hfsD in the wild-type strain had no substantial effect on fermentation products. Deletion of a single subunit, either A or subunit B (but not C or D), results in ethanol production near the theoretical maximum yield.