D-Galactose Catabolism in Penicillium Chrysogenum: Expression Analysis of the Structural Genes of the Leloir Pathway

Abstract

In this study, we analyzed the expression of the structural genes encoding the fve enzymes comprising the Leloir pathway of D-galactose catabolism in the industrial cell factory Penicillium chrysogenum on various carbon sources. The genome of P. chrysogenum contains a putative galactokinase gene at the annotated locus Pc13g10140, the product of which shows strong structural similarity to yeast galactokinase that was expressed on lactose and D-galactose only. The expression profle of the galactose-1-phosphate uridylyl transferase gene at annotated locus Pc15g00140 was essentially similar to that of galactokinase. This is in contrast to the results from other fungi such as Aspergillus nidulans, Trichoderma reesei and A. niger, where the ortholog galactokinase and galactose-1-phosphate uridylyl transferase genes were constitutively expressed. As for the UDP-galactose-4-epimerase encoding gene, fve candidates were identifed. We could not detect Pc16g12790, Pc21g12170 and Pc20g06140 expression on any of the carbon sources tested, while for the other two loci (Pc21g10370 and Pc18g01080) transcripts were clearly observed under all tested conditions. Like the 4-epimerase specifed at locus Pc21g10370, the other two structural Leloir pathway genes–UDP-glucose pyrophosphorylase (Pc21g12790) and phosphoglucomutase (Pc18g01390)–were expressed constitutively at high levels as can be expected from their indispensable function in fungal cell wall formation.

References

  1. 1.

    Alam, K., Kaminskyj, S. G. W. (2013) Aspergillus galactose metabolism is more complex than that of Saccharomyces: the story of GalDGAL7 and GalEGAL1. Botany 91, 467–477.

    CAS  Article  Google Scholar 

  2. 2.

    Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.

    CAS  Article  Google Scholar 

  3. 3.

    Beerens, K., Soetaert, W., Desmet, T. (2015) UDP-hexose 4-epimerases: a view on structure, mechanism and substrate specifcity. Carbohydrate Res. 414, 8–14.

    CAS  Article  Google Scholar 

  4. 4.

    Bhat, M. K. (2000) Cellulases and related enzymes in biotechnology. Biotechn. Adv. 18, 355–383.

    CAS  Article  Google Scholar 

  5. 5.

    Boschloo, J. G., Roberts, C. F. (1979) D-Galactose requiring mutants in Aspergillus nidulans lacking phosphoglucomutase. FEBS Lett. 104, 17–20.

    CAS  Article  Google Scholar 

  6. 6.

    Chiang, C., Knight, S. G. (1960) A new pathway of pentose metabolism. Biochem. Biophys. Res. Commun. 3, 554–559.

    CAS  Article  Google Scholar 

  7. 7.

    Christensen, U., Gruben, B. S., Madrid, S., Mulder, H., Nikolaev, I., de Vries, R. P. (2011) Unique regulatory mechanism for D-galactose utilization in Aspergillus nidulans. Appl. Environ. Microbiol. 77, 7084–7087.

    CAS  Article  Google Scholar 

  8. 8.

    Cragg, S. M., Beckham, G. T., Bruce, N. C., Bugg, T. D. H., Distel, D. L., Dupree, P., Etxabe, A. G., Goodell, B. S., Jellison, J., McGeehan, J. E., McQueen-Mason, S. J., Schnorr, K., Walton, P. H., Watts, J. E. M., Zimmer, M. (2015) Lignocellulose degradation mechanisms across the Tree of Life. Curr. Opin. Chem. Biol. 29, 108–119.

    CAS  Article  Google Scholar 

  9. 9.

    De Vries, R. P., Flipphi, M. J. A., Witteveen, C. F. B., Visser, J. (1994) Characterization of an Aspergillus nidulans L-arabitol dehydrogenase mutant. FEMS Microbiol. Lett. 123, 83–90.

    Article  Google Scholar 

  10. 10.

    De Vries, R. P., Visser, J. (2001) Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiol. Mol. Biol. Rev. 65, 497–522.

    Article  Google Scholar 

  11. 11.

    Edson, C. M., Brody, S. (1976) Biochemical and genetic studies on galactosamine metabolism in Neurospora crassa. J. Bacteriol. 126, 799–805.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    El-Ganiny, A. M., Sheoran, I., Sanders, D. A. R., Kaminskyj, S. G. W. (2010) Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture, hyphal morphogenesis, and asexual development. Fungal Genet. Biol. 47, 629–635.

    CAS  Article  Google Scholar 

  13. 13.

    Fekete, E., Karaffa, L., Sándor, E., Seiboth, B., Biró, S., Szentirmai, A., Kubicek, C. P. (2002) Regulation of formation of the intracellular β-galactosidase activity of Aspergillus nidulans. Arch. Microbiol. 179, 7–14.

    CAS  Article  Google Scholar 

  14. 14.

    Fekete, E., Karaffa, L., Sándor, E., Bányai, I., Seiboth, B., Gyémánt G., Sepsi, A, Szentirmai, A., Kubicek, C. P. (2004) The alternative D-galactose degrading pathway of Aspergillus nidulans proceeds via L-sorbose. Arch. Microbiol. 181, 35–44.

    CAS  Article  Google Scholar 

  15. 15.

    Fekete, E., De Vries, R. P., Seiboth, B., Van Kuyk, P. A., Sándor, E., Fekete, E., Metz, B., Kubicek, C. P., Karaffa, L. (2012) D-galactose uptake is nonfunctional in the conidiospores of Aspergillus niger. FEMS Microbiol. Lett. 329, 198–203.

    CAS  Article  Google Scholar 

  16. 16.

    Fekete, E., Karaffa, L., Seiboth, B., Fekete, E., Kubicek, C. P., Flipphi, M. (2012) Identifcation of a permease gene involved in lactose utilisation in Aspergillus nidulans. Fungal Genet. Biol. 49, 415–425.

    CAS  Article  Google Scholar 

  17. 17.

    Flipphi, M., Sun, J., Robellet, X., Karaffa, L., Fekete, E., Zeng, A. P., Kubicek, C. P. (2009) Biodiversity and evolution of primary carbon metabolism in Aspergillus nidulans and other Aspergillus spp. Fungal Genet. Biol. 46, S19–S44.

  18. 18.

    Free, S. J. (2013) Fungal cell wall organization and biosynthesis. Adv. Genet. 81, 33–82.

    CAS  Article  Google Scholar 

  19. 19.

    Frey, P. A. (1996) The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical confguration of a single carbon in galactose. FASEB J. 10, 461–470.

    CAS  Article  Google Scholar 

  20. 20.

    Haas, H., Angermayr, K., Zadra, I., Stöffer, G. (1997) Overexpression of nreB, a new GATA factor-encoding gene of Penicillium chrysogenum, leads to repression of the nitrate assimilatory gene cluster. J. Biol. Chem. 272, 22576–22582.

    CAS  Article  Google Scholar 

  21. 21.

    Houbraken, J., Frisvad, J. C., Samson, R. A. (2011) Fleming’s penicillin producing strain is not Penicillium chrysogenum but P. rubens. IMA Fungus 2, 87–95.

    Article  Google Scholar 

  22. 22.

    Houbraken, J., Samson, R. A. (2011) Phylogeny of Penicillium and the segregation of Trichocomaceae into three families. Stud. Mycol. 70, 1–51.

    CAS  Article  Google Scholar 

  23. 23.

    Hu, X., Robin, S., O’Connell, S., Walsh, G., Wall, J. G. (2010) Engineering of a fungal β-galactosidase to remove product inhibition by galactose. Appl. Microbiol. Biotechnol. 87, 1773–1782.

    CAS  Article  Google Scholar 

  24. 24.

    Jónás, Á., Fekete, E., Flipphi, M., Sándor, E., Jäger, S., Molnár, Á. P., Szentirmai, A., Karaffa, L. (2014) Extra- and intracellular lactose catabolism in Penicillium chrysogenum: Phylogenetic and expression analysis of the putative permease and hydrolase genes. J. Antibiot. 67, 489–497.

    Article  Google Scholar 

  25. 25.

    Jørgensen, H., Mørkeberg, A., Krogh, K. B., Olsson, L. (2004) Growth and enzyme production by three Penicillium species on monosaccharides. J. Biotechnol. 109, 295–299.

    Article  Google Scholar 

  26. 26.

    Lee, M. J., Gravelat, F. N., Cerone, R. P., Baptista, S. D., Campoli, P. V., Choe, S. I., Kravtsov, I., Vinogradov, E., Creuzenet, C., Liu, H., Berghuis, A. M., Latgé, J. P., Filler, S. G., Fontaine, T., Sheppard, D. C. (2014) Overlapping and distinct roles of Aspergillus fumigatus UDP-glucose 4-epimerases in galactose metabolism and the synthesis of galactose-containing cell wall polysac-charides. J. Biol. Chem. 289, 1243–1256.

    CAS  Article  Google Scholar 

  27. 27.

    Majumdar, S., Ghatak, J., Mukherji, S., Bhattacharjee, H., Bhaduri, A. (2004) UDPgalactose 4-epimerase from Saccharomyces cerevisiae: A bifunctional enzyme with aldose 1-epimerase activity. Eur. J. Biochem. 271, 753–759.

    CAS  Article  Google Scholar 

  28. 28.

    Milewski, S., Gabriel, I., Olchowy, J. (2006) Enzymes of UDP-GlcNAc biosynthesis in yeast. Yeast 23, 1–14.

    CAS  Article  Google Scholar 

  29. 29.

    Mojzita, D., Koivistoinen, O. M., Maaheimo, H., Penttilä, M., Ruohonen, L., Richard, P. (2012) Identifcation of the galactitol dehydrogenase, LadB, that is part of the oxido-reductive D-galactose catabolic pathway in Aspergillus niger. Fungal Genet. Biol. 49, 152–159.

    CAS  Article  Google Scholar 

  30. 30.

    Newbert, R. W., Barton, B., Greaves, P., Harper, J., Turner, G. (1997) Analysis of a commercially improved Penicillium chrysogenum strain series: Involvement of recombinogenic regions in amplifcation and deletion of the penicillin biosynthesis gene cluster. J. Ind. Microbiol. Biotechnol. 19, 18–27.

    CAS  Article  Google Scholar 

  31. 31.

    Ozcengiz, G., Demain, A. L. (2013) Recent advances in the biosynthesis of penicillins, cephalosporins and clavams and its regulation. Biotechnol. Adv. 31, 287–311.

    CAS  Article  Google Scholar 

  32. 32.

    Paul, B. C., El-Ganiny, A. M., Abbas, M., Kaminskyj, S. G., Dahms, T. E. (2011) Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy. Eukaryot. Cell 10, 646–653.

    CAS  Article  Google Scholar 

  33. 33.

    Pauly, M., Keegstra, K. (2010): Plant cell wall polymers as precursors for biofuels. Curr. Opin. Plant Biol. 13, 305–312.

    CAS  Article  Google Scholar 

  34. 34.

    Raper, K. B., Alexander, D. F., Coghill, R. D. (1944) Penicillin. II. Natural variation and penicillin production in Penicillium notatum and allied species. J. Bacteriol. 48, 639–659.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Roberts, C. F. (1970) Enzyme lesions in galactose non-utilising mutants of Aspergillus nidulans. Biochim. Biophys. Acta 201, 267–283.

    CAS  Article  Google Scholar 

  36. 36.

    Sakamoto, T., Ishimaru, M. (2013) Peculiarities and applications of galactanolytic enzymes that act on type I and II arabinogalactans. Appl. Microbiol. Biotechnol. 97, 5201–5213.

    CAS  Article  Google Scholar 

  37. 37.

    Sambrook, J., Russell, D. W. (2001) Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory, New York)

    Google Scholar 

  38. 38.

    Seiboth, B., Hofmann, G., Kubicek, C. P. (2002) Lactose metabolism and cellulase production in Hypocrea jecorina: the gal7 gene, encoding galactose-1-phosphate uridylyltransferase, is essential for growth on galactose but not for cellulase induction. Mol. Genet. Genomics 267, 124–132.

    CAS  Article  Google Scholar 

  39. 39.

    Seiboth, B., Hartl, L., Pail, M., Fekete, E., Karaffa, L., Kubicek, C. P. (2004) The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on D-galactose. Mol. Microbiol. 51, 1015–1025.

    CAS  Article  Google Scholar 

  40. 40.

    Wortman, J. R., Gilsenan, J. M., Joardar, V., Deegan, J., Clutterbuck, J., Andersen, M. R., Archer, D., Bencina, M., Braus, G., Coutinho, P., von Döhren, H., Doonan, J., Driessen, A. J., Durek, P., Espeso, E., Fekete, E., Flipphi, M., Garcia Estrada, C., Geysens, S., Goldman, G., de Groot, P. W., Hansen, K., Harris, S. D., Heinekamp, T., Helmstaedt, K., Henrissat, B., Hofmann, G., Homan, T., Horio, T., Horiuchi, H., James, S., Jones, M., Karaffa, L., Karányi, Z., Kato, M., Keller, N., Kelly, D. E., Kiel, J. A., Kim, J. M., van der Klei, I. J., Klis, F. M., Kovalchuk, A., Kraševec, N., Kubicek, C. P., Liu, B., MacCabe, A., Meyer, V., Mirabito, P., Miskei, M., Mos, M., Mullins, J., Nelson, D. R., Nielsen, J., Oakley, B. R., Osmani, S. A., Pakula, T., Paszewski, A., Paulsen, I., Pilsyk, S., Pócsi, I., Punt, P. J., Ram, A. F. J., Ren, Q., Robellet, X., Robson, G., Seiboth, B., van Solingen, P., Specht, T., Sun, J., Taheri-Talesh, N., Takeshita, N., Ussery, D., van Kuyk, P. A., Visser, H., van de Vondervoort, P. J., de Vries, R. P., Walton, J., Xiang, X., Xiong, Y., Zeng, A. P., Brandt, B. W., Cornell, M. J., van den Hondel, C. A. M. J. J., Visser, J., Oliver, S. G., Turner, G. (2009) The 2008 update of the Aspergillus nidulans genome annotation: a community effort. Fungal Genet. Biol. 46, S2–S13.

    CAS  Article  Google Scholar 

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Correspondence to Levente Karaffa.

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Jónás, Á., Fekete, E., Németh, Z. et al. D-Galactose Catabolism in Penicillium Chrysogenum: Expression Analysis of the Structural Genes of the Leloir Pathway. BIOLOGIA FUTURA 67, 318–332 (2016). https://doi.org/10.1556/018.67.2016.3.9

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Keywords

  • Penicillium chrysogenum
  • lactose
  • D-galactose
  • Leloir pathway