Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae.
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, ldhA and ldhB, 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 ldhA 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 ldhB were not detected when R. oryzae was grown on any of these sugars but were present when R. oryzae was grown on glycerol, ethanol, and lactate. I hypothesize that ldhB encodes a second NAD(+)-dependent LDH that is capable of converting L-lactate to pyruvate and is produced by cultures grown on these nonfermentable substrates. Both ldhA and ldhB restored fermentative growth to Escherichia coli (ldhA pfl) mutants so that they grew anaerobically and produced lactic acid.
Skory
C D
CD
Fermentation Biochemistry Research, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604-3902, USA. skorycd@mail.usda.gov
D000818:Animals; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012233:Rhizopus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D003470:Culture Media; D009243:NAD; D007770:L-Lactate Dehydrogenase