Native and modified lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880.
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.
Skory
Christopher D
CD
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
Ibrahim
Ashraf S
AS
D004271:DNA, Fungal / Q000302:isolation & purification; D005650:Fumarates / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000080864:Lactate Dehydrogenase 5; D019344:Lactic Acid / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis; D011995:Recombination, Genetic; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005650:Fumarates; D007527:Isoenzymes; D012333:RNA, Messenger; D019344:Lactic Acid; C032005:fumaric acid; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; C409667:lactate dehydrogenase 1; D009962:Orotate Phosphoribosyltransferase; D005947:Glucose