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Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/31/2000 Publication Date: N/A Citation: N/A Interpretive Summary: Lactic acid has long been used by the food industry as an additive for preservation, flavor, and acidity. Recently, it has gained popularity for the manufacture of environmentally friendly products which include the biodegradable plastic, poly-lactic acid (PLA), and the chlorine-free solvent, ethyl lactate. Lactic acid is typically made by microorganisms that are able to convert or ferment sugars obtained from agricultural crops, such as corn. The lactic acid market for the U.S. is currently about 50,000 tons/yr and could increase substantially if the market for PLA develops as expected. Fermentations with the fungus Rhizopus are often preferred because the quality of the final product is superior to that obtained by bacterial fermentations. Furthermore, this organism produces lactic acid in the chemical form that is more desirable for making PLA. We have isolated and characterized two genes from Rhizopus that are involved in the production of lactic acid. This is the first description of these kinds of genes ever found in a fungus. The same type of genes have been extensively studied in animal, plant, and bacteria, so this work is expected to fill a significant void. Our work demonstrates how these genes function in Rhizopus to convert sugars into lactic acid. Additionally, this discovery will allow us to modify and re-introduce the genes back into Rhizopus in an attempt to improve the rate at which the organism produces lactic acid. Technical Abstract: Two genes, ldhA and ldhB, coding for NAD+ dependent L-lactate dehydrogenase (LDH, EC 1.1.1.27) have been isolated from the fungus Rhizopus oryzae. Sequence comparisons from genomic DNA and cDNA show that there are no introns present in either gene. The ldhA and ldhB are very similar with greater than 90% identity. However, they are very distinct from other lactate dehydrogenase with the greatest similarity being shared with two LDH in the Thermus/Deinococcus group with only a 42% similarity in protein sequence. Amino acid sequence analysis of the LdhA protein isolated from R. oryzae during lactic acid production confirmed that the ldhA codes for a 36 Kda protein involved in the conversion of pyruvate to lactate. Production of LdhA was highest in glucose containing medium, followed by xylose and trehalose, which are all capable of being fermented to lactic acid. Transcript from ldhB could not be detected in any of these sugars but was present when R. oryzae was grown in glycerol, ethanol, and lactate Zymogram analysis of protein extracts from these cultures demonstrated the presence of an NAD+ dependent LDH capable of converting L-lactate to pyruvate, suggesting a relationship to ldhB. Both genes were capable of complementing Escherichia coli (ldhA, pfl) mutants, such that they could grow anaerobically and produce lactic acid. |
