Skip to main content
Log in

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

  • Research Article
  • Published:
Current Genetics Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Abe A, Sone T, Sujaya IN, Saito K, Oda Y, Asano K, Tomita F (2003) rDNA ITS sequence of Rhizopus oryzae: its application to classification and identification of lactic acid producers. Biosci Biotechnol Biochem 67:1725–1731

    Article  PubMed  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    PubMed  CAS  Google Scholar 

  • Babiychuk E, Kushnir S, Belles-Boix E, Van MM, Inze D (1995) Arabidopsis thaliana NADPH oxidoreductase homologs confer tolerance of yeasts toward the thiol-oxidizing drug diamide. J Biol Chem 270:26224–26231

    Article  PubMed  CAS  Google Scholar 

  • Bai DM, Zhao XM, Li XG, Xu SM (2004) Strain improvement of Rhizopus oryzae for over-production of L(+)-lactic acid and metabolic flux analysis of mutants. Biochem Eng J 18:41–48

    Article  CAS  Google Scholar 

  • Cameron AD, Roper DI, Moreton KM, Muirhead H, Holbrook JJ, Wigley DB (1994) 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 238:615–625

    Article  PubMed  CAS  Google Scholar 

  • Clarke AR, Wigley DB, Barstow DA, Chia WN, Atkinson T, Holbrook JJ (1987) A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure. Biochim Biophys Acta 913:72–80

    PubMed  CAS  Google Scholar 

  • de Arriaga D, Soler J, Cadenas E (1982) Influence of pH on the allosteric properties of lactate dehydrogenase activity of Phycomyces blakesleeanus. Biochem J 203:393–400

    PubMed  Google Scholar 

  • Ehrlich F (1911) Über die bildung von fumarsäure durch schimmelpilze. Ber Dtsch Chem Ges 44:3737–3742

    Google Scholar 

  • Eventoff W, Rossmann MG, Taylor SS, Torff HJ, Meyer H, Keil W, Kiltz HH (1977) Structural adaptations of lactate dehydrogenase isozymes. Proc Natl Acad Sci USA 74:2677–2681

    Article  PubMed  CAS  Google Scholar 

  • Foster JW, Waksman SA (1939) The production of fumaric acid by molds belonging to the genus Rhizopus. J Am Chem Soc 61:127–135

    Article  CAS  Google Scholar 

  • Garvie EI (1980) Bacterial lactate dehydrogenases. Microbiol Rev 44:106–139

    PubMed  CAS  Google Scholar 

  • Ge CM, Gu SBZXH, Yao JM, Pan RR, Yu ZL (2004) Breeding of L(+)-lactic acid producing strain by low-energy ion implantation. J Microbiol Biotechnol 14:363–366

    CAS  Google Scholar 

  • Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326

    Article  PubMed  CAS  Google Scholar 

  • Henriksen A, Aghajari N, Jensen KF, Gajhede M (1996) A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase. Biochemistry 35:3803–3809

    Article  PubMed  CAS  Google Scholar 

  • Ibrahim AS, Avanessian V, Spellberg B, Edwards JE Jr (2003) Liposomal amphotericin B, and not amphotericin B deoxycholate, improves survival of diabetic mice infected with Rhizopus oryzae. Antimicrob Agents Chemother 47:3343–3344

    Article  PubMed  CAS  Google Scholar 

  • Kenealy W, Zaady E, DuPreez JC, Stieglitz B, Goldberg I (1986) Biochemical aspects of fumaric acid accumulation by Rhizopus arrhizus. Appl Environ Microbiol 52:128–133

    PubMed  CAS  Google Scholar 

  • Lockwood LB, Ward GE, May OE (1936) The physiology of Rhizopus oryzae. J Agric Res 53:849–857

    CAS  Google Scholar 

  • Longacre A, Reimers JM, Gannon JE, Wright BE (1997) Flux analysis of glucose metabolism in Rhizopus oryzae for the purpose of increasing lactate yields. Fungal Genet Biol 21:30–39

    Article  PubMed  CAS  Google Scholar 

  • Magnuson JK, Lasure LL (2004) Organic acid production by filamentous fungi. In: Tkacz JS, Lange L (eds) Advances in fungal biotechnology for industry, agriculture, and medicine. Springer, Berlin, Heidelberg, New York, pp 307–340

    Google Scholar 

  • Margulies M, Vishniac W (1961) Dissimilation of glucose by the MX strain of Rhizopus. J Bacteriol 81:1–9

    Article  PubMed  CAS  Google Scholar 

  • Matsuzawa H, Machida M, Kunai K, Ito Y, Ohta T (1988) Identification of an allosteric site residue of a fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase of Thermus caldophilus GK24: production of a non-allosteric form by protein engineering. FEBS Lett 233:375–378

    Article  CAS  Google Scholar 

  • Michielse CB, Salim K, Ragas P, Ram AF, Kudla B, Jarry B, Punt PJ, van den Hondel CA (2004) Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer. Mol Genet Genomics 271:499–510

    Article  PubMed  CAS  Google Scholar 

  • Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J (2000) Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng 2:69–77

    Article  PubMed  CAS  Google Scholar 

  • Nordstöm K (1968) Yeast growth and glycerol formation II. Carbon and redox balances. J Inst Brew 74:429–432

    Google Scholar 

  • Obayashi A, Yorifuji H, Yamagata T, Ijichi T, Kanie M (1966) Respiration in organic acid forming molds: Part I. Purification of cytochrome c, coenzyme Q and L-lactic dehydrogenase from lactate forming Rhizopus oryzae. Agric Biol Chem 30:717–724

    CAS  Google Scholar 

  • Osmani SA, Scrutton MC (1985) The sub-cellular localisation and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus. Eur J Biochem 147:119–128

    Article  PubMed  CAS  Google Scholar 

  • Ozturk DH, Dorfman RH, Scapin G, Sacchettini JC, Grubmeyer C (1995) Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase. Biochemistry 34:10755–10763

    Article  PubMed  CAS  Google Scholar 

  • Page JP, Munagala NR, Wang CC (1999) Point mutations in the guanine phosphoribosyltransferase from Giardia lamblia modulate pyrophosphate binding and enzyme catalysis. Eur J Biochem 259:565–571

    Article  PubMed  CAS  Google Scholar 

  • Peleg Y, Battat E, Scrutton MC, Goldberg I (1989) Isozyme pattern and subcellular localisation of enzyme involved in fumaric acid accumulation by Rhizopus oryzae. Appl Microbiol Biotechnol 32:334–339

    Article  CAS  Google Scholar 

  • Pritchard GG (1973) Factors affecting the activity and synthesis of NAD dependent lactate dehydrogenase in Rhizopus oryzae. J Gen Microbiol 78:125–137

    CAS  Google Scholar 

  • Ribes JA, Vanover-Sams CL, Baker DJ (2000) Zygomycetes in human disease. Clin Microbiol Rev 13:236–301

    Article  PubMed  CAS  Google Scholar 

  • Saito K (1911) Ein Beispiel von Milchsäurebildung durch schimmelpilze. Centralbl Bakt II 29:289–290

    Google Scholar 

  • Saito K, Saito A, Ohnishi M, Oda Y (2004) Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes. Arch Microbiol 182:30–36

    Article  PubMed  CAS  Google Scholar 

  • Scapin G, Ozturk DH, Grubmeyer C, Sacchettini JC (1995) The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate. Biochemistry 34:10744–10754

    Article  PubMed  CAS  Google Scholar 

  • Schmitt ME, Brown TA, Trumpower BL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18:3091–3092

    Article  PubMed  CAS  Google Scholar 

  • Skory CD (2000) Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae. Appl Environ Microbiol 66:2343–2348

    Article  PubMed  CAS  Google Scholar 

  • Skory CD (2002) Homologous recombination and double-strand break repair in the transformation of Rhizopus oryzae. Mol Genet Genomics 268:397–406

    Article  PubMed  CAS  Google Scholar 

  • Skory CD (2003) Induction of Rhizopus oryzae pyruvate decarboxylase genes. Curr Microbiol 47:59–64

    Article  PubMed  CAS  Google Scholar 

  • Skory CD (2004a) Lactic acid production by Rhizopus oryzae transformants with modified lactate dehydrogenase activity. Appl Microbiol Biotechnol 64:237–242

    Article  PubMed  CAS  Google Scholar 

  • Skory CD (2004b) Repair of plasmid DNA used for transformation of Rhizopus oryzae by gene conversion. Curr Genet 45:302–310

    Article  PubMed  CAS  Google Scholar 

  • Skory CD (2005) Inhibition of non-homologous end joining and integration of DNA upon transformation of Rhizopus oryzae. Mol Genet Genomics 274:373–383

    Article  PubMed  CAS  Google Scholar 

  • Skory CD, Freer SN, Bothast RJ (1998) Production of L-lactic acid by Rhizopus oryzae under oxygen limiting conditions. Biotechnol Lett 20:191–194

    Article  CAS  Google Scholar 

  • Spellberg B, Fu Y, Edwards JE Jr, Ibrahim AS (2005) Combination therapy with amphotericin B lipid complex and caspofungin acetate of disseminated zygomycosis in diabetic ketoacidotic mice. Antimicrob Agents Chemother 49:830–832

    Article  PubMed  CAS  Google Scholar 

  • van Dijken JP, Scheffers WA (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol Rev 32:199–225

    Google Scholar 

  • Velayos A, Alvarez MI, Eslava AP, Iturriaga EA (1998) Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides. Mol Gen Genet 260:251–260

    Article  PubMed  CAS  Google Scholar 

  • Waksman SA, Hutchings IJ (1937) Lactic acid production by species of Rhizopus. J Am Chem Soc 59:545–547

    Article  CAS  Google Scholar 

  • Wright BE, Longacre A, Reimers J (1996) Models of metabolism in Rhizopus oryzae. J Theor Biol 182:453–457

    Article  PubMed  CAS  Google Scholar 

  • Yu R-C, Hang YD (1991) Purification and characterization of NAD-dependent lactate dehydrogenase from Rhizopus oryzae. Food Chem 41:219–225

    Article  CAS  Google Scholar 

Download references

Acknowledgments

ASI is supported by Public Health Service grants RO1 AI063503-01A2 and R21 AI064716-01A2.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Christopher D. Skory.

Additional information

Communicated by U. Kück.

Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Skory, C.D., Ibrahim, A.S. Native and modified lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880. Curr Genet 52, 23–33 (2007). https://doi.org/10.1007/s00294-007-0135-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-007-0135-0

Keywords

Navigation