Cloning, expression and complete nucleotide sequence of the Bacillus stearothermophilus L-lactate dehydrogenase gene

The structural gene for L-lactate dehydrogenase (LDH; EC 1.1.1.27) from Bacillus stearothermophilus NCA 1503 has been cloned in Escherichia coli and its complete nucleotide sequence determined. The predicted amino acid (aa) sequence of the LDH enzyme agrees with the previously determined aa sequence except to three positions: aa 125 and 126, Ser-Glu, are inverted whilst His at position 130 has been replaced by Ser in our sequence. The lct gene consists of an open reading frame (ORF) commencing from the ATG start codon of 951 bp followed by a TGA stop codon. Upstream from the start codon is a strong (delta G = -14.4 kcal) Shine-Dalgarno (SD) sequence, a feature typical of Gram-positive ribosome binding sites. Putative RNA polymerase recognition signals (-35 and -10 regions) have been identified upstream from the lct structural gene but there are no structures resembling Rho-independent transcription termination signals downstream from the TGA stop codon. Two further ORFs, preceded by SD sequences, are present downstream from the lct gene. Thus the lct gene may constitute the first gene of an operon. Subclones of the lct gene have been constructed in the expression plasmid pKK223-3 and the LDH enzyme produced in soluble form at levels of up to 36% of the E. coli soluble cell protein.