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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.

Author information

1
Bioproducts and Biocatalysis Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA. Chris.Skory@ars.usda.gov

Abstract

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.

PMID:
17551728
DOI:
10.1007/s00294-007-0135-0
[Indexed for MEDLINE]

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