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Curr Genet. 2007 Jul;52(1):23-33. Epub 2007 Jun 6.

Native and modified lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880.

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Bioproducts and Biocatalysis Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA.


Rhizopus oryzae is 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 99-880. LdhB enzyme purified from gene clones isolated from the Type-I isolate R. oryzae NRRL 395 and the Type-II isolate R. oryzae 99-880 each showed reductive LDH activity (pyruvate to lactate), while no oxidative LDH activity (lactate to pyruvate) was detected in either preparation. A transformation system was then developed for the first time with R. oryzae 99-880, 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 and 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.

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