Expression cloning of the nox, mdh and ldh genes from Thermus species encoding NADH oxidase, malate dehydrogenase and lactate dehydrogenase

The Thermus thermophilus HB8 mdh and ldh genes and the T. aquaticus EP00276 nox and mdh genes encoding the biotechnologically important enzymes NADH oxidase (EC 1.6.99.3), malate dehydrogenase (EC 1.1.1.37) and lactate dehydrogenase (EC 1.1.1.27) were cloned on the basis of known sequences from related species using the polymerase chain reaction. The nox and mdh genes were directly placed under the control of regulatory expression elements from Escherichia coli. When the 5'-portions of the re-cloned nox gene and the mdh gene of T. thermophilus HB8 were simultaneously altered, enzyme yields of 18-42% of the total soluble cellular protein were obtained as compared to 2-6% obtained from the unchanged genes. The high overproduction level upon the alterations can be explained by the occurrence of additional potential base pairs between nucleotides in the mRNA downstream of the start codon ('downstream box') and the 16S rRNA. An 'universal translation initiation sequence' providing such strong interactions may be of general use for high overproduction levels.