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Title: Native and Modified Lactate Dehydrogenase Expression in a Fumaric Acid Producing isolate Rhizopus oryzae 99-880

Author
item Skory, Christopher - Chris
item IBRAHIM, ASHRAF - UCLA MEDICAL CENTER

Submitted to: Current Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/8/2007
Publication Date: 6/6/2007
Citation: Skory, C.D., Ibrahim, A.S. 2007. Native and modified lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880. Current Genetics. 52:23-33.

Interpretive Summary: The fungus Rhizopus is frequently used to convert, or ferment sugars obtained from agricultural crops to lactic acid. This natural product has long been utilized by the food industry as an additive for preservation, flavor, and acidity. Additionally, it is used for the manufacture of environmentally friendly products which include the biodegradable plastic, poly-lactic acid (PLA), and the chlorine-free solvent, ethyl lactate. In order to allow the market potential of lactic acid to continue expanding at the current rapid pace, it is important that the production costs are minimized by the development of new and improved technologies. In this work, we reveal some of the genetic mechanisms involved in the production of lactic acid and provide additional techniques to improve current fermentation technology. The results of this study will allow new strategies to be developed for the industrial utilization of this valuable organism, thereby benefiting the agricultural grower and ultimately the consumer.

Technical Abstract: Rhizopus oryzae is a filamentous fungus that is of broad importance to the industrial, agricultural, and medical community. R. oryzae can be subdivided into two groups based on genetic and phenotypic differences. Type-I isolates accumulate primarily lactic acid when grown in the presence of a fermentable carbon source and contain two lactate dehydrogenase genes, ldhA and ldhB. Type-II isolates synthesize predominantly fumaric acid and only have an ldhB gene. In this study, we determined that ldhB transcript is only minimally expressed in the Type-II isolate R. oryzae RA99-880. LdhB enzyme purified from gene clones isolated from the Type-I isolate R. oryzae NRRL 395 and the Type-II isolate R. oryzae RA99-880 each showed reductive LDH activity (pyruvate to lactate) of 21.4 U/mg protein and 9.1 U/mg protein, respectively, while no oxidative LDH activity (lactate to pyruvate) was detected in either preparation. A transformation system was then developed with R. oryzae RA99-880, using a uracil auxotrophic isolate that could be complemented with an orotate phosphoribosyltransferase gene, pyrF, isolated in this study. Transformation of this Type-II isolate with the ldhA gene from R. oryzae NRRL 395 resulted in reductive LDH activity between 1.0-1.8 U/mg total protein. Additionally, transformed isolates grown with glucose accumulated up to 27 g lactic acid/L with a concurrent decrease in fumaric acid, ethanol, and glycerol compared with the untransformed and vector-transformed control strains. These results demonstrate that the Type-II isolate R. oryzae RA99-880 has the ability to express an ldhA gene using the native promoter. This enzyme effectively competes for available pyruvate, allowing the strain to synthesize and export lactic acid in amounts that are comparable to a Type-I isolate.