Isolation and Expression of Lactate Dehydrogenase Genes from Rhizopus olyzae

Rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. In this study I cloned two genes, IdhA and IdhB, which code for NAD+-dependent L-lactate dehydrogenases (LDH) (EC 1.1.1.27), from a lactic acid-producing strain of R. oryzae. These genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. This is the first description of ldh genes in a fungus, and sequence comparisons revealed that these genes are distinct from previously isolated prokaryotic and eukaryotic ldh genes. Protein sequencing of the LDH isolated from R. oryzae during lactic acid production confirmed that IdhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of LdhA was greatest when glucose was the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid. Transcripts from IdhB were not detected when R. oryzae was grmln on any of these sugars but were present when R. O1yzae was grown on glycerol, ethanol, and lactate. I hypothesize that IdhB encodes a second NAD+-dependent LDH that is capable of converting L-lactate to pyruvate and is produced by cultures grmlTI on these nonfermentable substrates. Both IdhA and IdhB restored fermentative growth to Escherichia coli (ldhA pfi) mutants so that they grew anaerobically and produced lactic acid.