Native and Modified Lactate Dehydrogenase Expression in a Fumaric Acid Producing Isolate Rhizopus Oryzae 99-880

Overview

Journal Curr Genet

Specialty Genetics

Date 2007 Jun 7

PMID 17551728

Citations 14

Authors

Christopher D Skory

Ashraf S Ibrahim

Affiliations

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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.

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References

GARVIE E . Bacterial lactate dehydrogenases. Microbiol Rev. 1980; 44(1):106-39. PMC: 373236. DOI: 10.1128/mr.44.1.106-139.1980. View

Kenealy W, Zaady E, du Preez J, Stieglitz B, Goldberg I . Biochemical Aspects of Fumaric Acid Accumulation by Rhizopus arrhizus. Appl Environ Microbiol. 1986; 52(1):128-33. PMC: 203406. DOI: 10.1128/aem.52.1.128-133.1986. View

Babiychuk E, Kushnir S, Van Montagu M, Inze D . Arabidopsis thaliana NADPH oxidoreductase homologs confer tolerance of yeasts toward the thiol-oxidizing drug diamide. J Biol Chem. 1995; 270(44):26224-31. DOI: 10.1074/jbc.270.44.26224. View

Cameron A, Roper D, Moreton K, MUIRHEAD H, HOLBROOK J, Wigley D . Allosteric activation in Bacillus stearothermophilus lactate dehydrogenase investigated by an X-ray crystallographic analysis of a mutant designed to prevent tetramerization of the enzyme. J Mol Biol. 1994; 238(4):615-25. DOI: 10.1006/jmbi.1994.1318. View

Wright B, LONGACRE A, Reimers J . Models of metabolism in Rhizopus oryzae. J Theor Biol. 1996; 182(3):453-7. DOI: 10.1006/jtbi.1996.0186. View

Skory C . Lactic acid production by Rhizopus oryzae transformants with modified lactate dehydrogenase activity. Appl Microbiol Biotechnol. 2003; 64(2):237-42. DOI: 10.1007/s00253-003-1480-7. View

Skory C . Inhibition of non-homologous end joining and integration of DNA upon transformation of Rhizopus oryzae. Mol Genet Genomics. 2005; 274(4):373-83. DOI: 10.1007/s00438-005-0028-1. View

Michielse C, Salim K, Ragas P, Ram A, Kudla B, Jarry B . Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer. Mol Genet Genomics. 2004; 271(4):499-510. DOI: 10.1007/s00438-004-1003-y. View

Ribes J, Baker D . Zygomycetes in human disease. Clin Microbiol Rev. 2001; 13(2):236-301. PMC: 100153. DOI: 10.1128/CMR.13.2.236. View

10.

Ibrahim A, Avanessian V, Spellberg B, Edwards Jr J . Liposomal amphotericin B, and not amphotericin B deoxycholate, improves survival of diabetic mice infected with Rhizopus oryzae. Antimicrob Agents Chemother. 2003; 47(10):3343-4. PMC: 201164. DOI: 10.1128/AAC.47.10.3343-3344.2003. View

11.

Skory C . Homologous recombination and double-strand break repair in the transformation of Rhizopus oryzae. Mol Genet Genomics. 2002; 268(3):397-406. DOI: 10.1007/s00438-002-0760-8. View

12.

Scapin G, Ozturk D, Grubmeyer C, Sacchettini J . The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate. Biochemistry. 1995; 34(34):10744-54. DOI: 10.1021/bi00034a006. View

13.

Eventoff W, Rossmann M, Taylor S, Torff H, Meyer H, KEIL W . Structural adaptations of lactate dehydrogenase isozymes. Proc Natl Acad Sci U S A. 1977; 74(7):2677-81. PMC: 431242. DOI: 10.1073/pnas.74.7.2677. View

14.

Clarke A, Wigley D, Barstow D, Chia W, Atkinson T, HOLBROOK J . A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure. Biochim Biophys Acta. 1987; 913(1):72-80. DOI: 10.1016/0167-4838(87)90234-2. View

15.

Saito K, Saito A, Ohnishi M, Oda Y . Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes. Arch Microbiol. 2004; 182(1):30-6. DOI: 10.1007/s00203-004-0691-3. View

16.

Henriksen A, Aghajari N, Jensen K, Gajhede M . A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase. Biochemistry. 1996; 35(12):3803-9. DOI: 10.1021/bi952226y. View

17.

Skory C . Repair of plasmid DNA used for transformation of Rhizopus oryzae by gene conversion. Curr Genet. 2004; 45(5):302-10. DOI: 10.1007/s00294-004-0494-8. View

18.

Ozturk D, Dorfman R, Scapin G, Sacchettini J, Grubmeyer C . Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase. Biochemistry. 1995; 34(34):10755-63. DOI: 10.1021/bi00034a007. View

19.

Gill S, von Hippel P . Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989; 182(2):319-26. DOI: 10.1016/0003-2697(89)90602-7. View

20.

Altschul S, Gish W, Miller W, Myers E, Lipman D . Basic local alignment search tool. J Mol Biol. 1990; 215(3):403-10. DOI: 10.1016/S0022-2836(05)80360-2. View