Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae

Abstract

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.

FIG. 1

Relationship of LDH subunits from numerous hosts. A most parsimonious tree for 26 LDH amino acid sequences is shown. Levels of amino acid substitution are expressed as percentages (bar = 10%). Most of the nodes have levels of bootstrap support of 99 to 100%; the only exceptions are the nodes labeled a (78 to 80%) or b (44 to 60%). Data for Streptococcus thermophilus (13), Streptococcus bovis (37), Streptococcus mutans (5), Lactococcus lactis (17), Lactobacillus plantarum (30), Lactobacillus sake (accession no. U26688), Lactobacillus casei (15), Bacillus megaterium (34), Bacillus stearothermophilus (40), Bacillus caldolyticus (40), Thermus aquaticus (23), Deinococcus radiodurans (21), corn (9), rice (16), tomato (8), human A (32), human B (27), pigs A and B (31), bovine A (12), mouse A, chicken A (11), dogfish A (28), and lamprey (29) have been published previously.

FIG. 2

Northern analysis of ldh transcript accumulation after glycerol-peptone-grown R. oryzae was transferred to media containing new carbon sources and incubated for 5.5 h. Lane 1, glucose; lane 2, xylose; lane 3, trehalose; lane 4, peptone; lane 5, glycerol; lane 6, ethanol; lane 7, lactate; lane 8, glycerol-peptone. The gel used for transfer to the membrane is shown at the bottom. The locations of molecular weight standards (in kilobases) are indicated on the left. Due to the similarity of ldhA and ldhB, the labeled ldhA riboprobe detected both transcripts.

FIG. 3

RPA for ldh accumulation in a bubble column apparatus. RNA was purified at various times during fermentation and hybridized to the ldhA riboprobe for RPA analysis. The conditions used for the analysis were such that the probe protected both ldhA and ldhB. The control was yeast RNA hybridized to the probe. The numbers at the top indicate the times (in hours) that the RNA were obtained.