DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases

Total DNA of a population of uncultured organisms was extracted from soil s amples, and by using PCR methods, the genes encoding two different 2,5-dike to-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primer s based on published sequence information gave internal gene fragments homo logous to known DKGRs. Nested primers specific for the internal fragments w ere combined with random primers to amplify flanking gene fragments from th e environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers w ere used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catal yzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exh ibited greater than 20-fold-higher k(cat)/K-m values than those previously determined, primarily as a result of better binding of substrate. The K-m v alues for the two new reductases were 57 and 67 muM, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as wel l as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reducta ses use only NADPH. In addition, one of the new reductases was more thermos table than known DKGRs.