Figure 1

Change from DEH to DRP-1 and DRP-2. (A) DEH (1% w/v) changed to DRP-1 and DRP-2 after incubation with 50 mM Tris-HCl (pH 7.5). DEH (1% w/v) was incubated at 30 °C for 30 h in the presence (lane 2) or absence (lane 3) of 50 mM Tris-HCl (pH 7.5), developed, and detected using the sulfate method (left) and thiobarbituric acid (right) methods. DEH was prepared without 50 mM Tris-HCl (pH 7.5) as described previously¹³. Lane 1, 1% (w/v) DEH alone. Positions of DEH, DRP-1, and DRP-2 are indicated by arrows. (B) The prototrophic bioengineered S. cerevisiae DEH++ (MK6286) utilized DEH but not DRP-1 or DRP-2. The MK6286 strain was cultivated in 1.0 mL of DEH + Asn (5 mM) (closed symbols) or DRP-1 + Asn (5 mM) (open symbols) media (upper panel). These cultures were obtained, analyzed by TLC, and DEH was detected using the thiobarbituric acid method (lower panel). DEH corresponds to 1% DEH. The density of DEH decreased gradually in a time-dependent manner, whereas the densities of DRP-1 and DRP-2 did not decrease. We failed to determine the density of DEH because the signals were weak. (C) DEH was enzymatically reduced to 2-keto-3-deoxy-d-gluconate using purified DEH reductase (A1-R′). The reaction was conducted in the presence (+) or absence (−) of A1-R′, as described in Methods. Samples were analyzed by TLC, and DEH was detected using the sulfate method. KDG, 2-keto-3-deoxy-d-gluconate.