1887

Abstract

The filamentous ascomycete is the only industrial producer of the β-lactam antibiotic cephalosporin C. Synthesis of all β-lactam antibiotics starts with the three amino acids -α-aminoadipic acid, -cysteine and -valine condensing to form the δ-(-α-aminoadipyl)--cysteinyl--valine tripeptide. The availability of building blocks is essential in every biosynthetic process and is therefore one of the most important parameters required for optimal biosynthetic production. Synthesis of -cysteine is feasible by various biosynthetic pathways in all euascomycetes, and sequencing of the genome has shown that a full set of sulfur-metabolizing genes is present. In principle, two pathways are effective: an autotrophic one, where the sulfur atom is taken from assimilated sulfide to synthesize either -cysteine or -homocysteine, and a reverse transsulfuration pathway, where -methionine is the sulfur donor. Previous research with production strains has focused on reverse transsulfuration, and concluded that both -methionine and reverse transsulfuration are essential for high-level cephalosporin C synthesis. Here, we conducted molecular genetic analysis with A3/2, another production strain, to investigate the autotrophic pathway. Strains lacking either cysteine synthase or homocysteine synthase, enzymes of the autotrophic pathway, are still autotrophic for sulfur. However, deletion of both genes results in sulfur amino acid auxotrophic mutants exhibiting delayed biomass production and drastically reduced cephalosporin C synthesis. Furthermore, both single- and double-deletion strains are more sensitive to oxidative stress and form fewer arthrospores. Our findings provide evidence that autotrophic sulfur assimilation is essential for growth and cephalosporin C biosynthesis in production strain A3/2 from .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000474
2017-06-01
2019-12-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/163/6/817.html?itemId=/content/journal/micro/10.1099/mic.0.000474&mimeType=html&fmt=ahah

References

  1. Demain AL. Antibiotics: natural products essential to human health. Med Res Rev 2009;29:821–842 [CrossRef][PubMed]
    [Google Scholar]
  2. Ozcengiz G, Demain AL. Recent advances in the biosynthesis of penicillins, cephalosporins and clavams and its regulation. Biotechnol Adv 2013;31:287–311 [CrossRef][PubMed]
    [Google Scholar]
  3. Barton AB, Kaback DB, Clark MW, Keng T, Ouellette BF et al. Physical localization of yeast CYS3, a gene whose product resembles the rat γ-cystathionase and Escherichia coli cystathionine γ-synthase enzymes. Yeast 1993;9:363–369 [CrossRef][PubMed]
    [Google Scholar]
  4. Cherest H, Thomas D, Surdin-Kerjan Y. Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulfuration pathway which has been built up by enzyme recruitment. J Bacteriol 1993;175:5366–5374 [CrossRef][PubMed]
    [Google Scholar]
  5. Segel IH, Johnson MJ. Intermediates in inorganic sulfate utilization by Penicillium chrysogenum. Arch Biochem Biophys 1963;103:216–226 [CrossRef][PubMed]
    [Google Scholar]
  6. Drew SW, Demain AL. Production of cephalosporin C by single and double sulfur auxotrophic mutants of Cephalosporium acremonium. Antimicrob Agents Chemother 1975;8:5–10 [CrossRef][PubMed]
    [Google Scholar]
  7. Drew SW, Demain AL. Stimulation of cephalosporin production by methionine peptides in a mutant blocked in reverse transsulfuration. J Antibiot 1975;28:889–895 [CrossRef][PubMed]
    [Google Scholar]
  8. Döbeli H, Nüesch J. Regulatory properties of O-acetyl-L-serine sulfhydrylase of Cephalosporium acremonium: evidence of an isoenzyme and its importance in cephalosporin C biosynthesis. Antimicrob Agents Chemother 1980;18:111–117 [CrossRef][PubMed]
    [Google Scholar]
  9. Martín JF, Demain AL. Unraveling the methionine-cephalosporin puzzle in Acremonium chrysogenum. Trends Biotechnol 2002;20:502–507 [CrossRef][PubMed]
    [Google Scholar]
  10. Sambrook J, Russell DW. Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001
    [Google Scholar]
  11. Bullock WO, Fernandez JM, Short JM. XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. Biotechniques 1987;5:376–379
    [Google Scholar]
  12. Colot HV, Park G, Turner GE, Ringelberg C, Crew CM et al. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci USA 2006;103:10352–10357 [CrossRef][PubMed]
    [Google Scholar]
  13. Becker DM, Lundblad V. Introduction of DNA into yeast cells. Curr Protoc Mol Biol 2001;Chapter 13:Unit 13.7 [CrossRef][PubMed]
    [Google Scholar]
  14. Walz M, Kück U. Polymorphic karyotypes in related Acremonium strains. Curr Genet 1991;19:73–76 [CrossRef][PubMed]
    [Google Scholar]
  15. Gsaller F, Blatzer M, Abt B, Schrettl M, Lindner H et al. The first promoter for conditional gene expression in Acremonium chrysogenum: iron starvation-inducible mir1(P). J Biotechnol 2013;163:77–80 [CrossRef][PubMed]
    [Google Scholar]
  16. Radzio R, Kück U. Efficient synthesis of the blood-coagulation inhibitor hirudin in the filamentous fungus Acremonium chrysogenum. Appl Microbiol Biotechnol 1997;48:58–65 [CrossRef][PubMed]
    [Google Scholar]
  17. Walz M, Kück U. Targeted integration into the Acremonium chrysogenum genome: disruption of the pcbC gene. Curr Genet 1993;24:421–427 [CrossRef][PubMed]
    [Google Scholar]
  18. Dreyer J, Eichhorn H, Friedlin E, Kürnsteiner H, Kück U. A homologue of the Aspergillus velvet gene regulates both cephalosporin C biosynthesis and hyphal fragmentation in Acremonium chrysogenum. Appl Environ Microbiol 2007;73:3412–3422 [CrossRef][PubMed]
    [Google Scholar]
  19. Bloemendal S, Löper D, Terfehr D, Kopke K, Kluge J et al. Tools for advanced and targeted genetic manipulation of the β-lactam antibiotic producer Acremonium chrysogenum. J Biotechnol 2014;169:51–62 [CrossRef][PubMed]
    [Google Scholar]
  20. Hoff B, Schmitt EK, Kück U. CPCR1, but not its interacting transcription factor AcFKH1, controls fungal arthrospore formation in Acremonium chrysogenum. Mol Microbiol 2005;56:1220–1233 [CrossRef][PubMed]
    [Google Scholar]
  21. Kopke K, Hoff B, Bloemendal S, Katschorowski A, Kamerewerd J et al. Members of the Penicillium chrysogenum velvet complex play functionally opposing roles in the regulation of penicillin biosynthesis and conidiation. Eukaryot Cell 2013;12:299–310 [CrossRef][PubMed]
    [Google Scholar]
  22. Brzywczy J, Paszewski A. Role of O-acetylhomoserine sulfhydrylase in sulfur amino acid synthesis in various yeasts. Yeast 1993;9:1335–1342 [CrossRef][PubMed]
    [Google Scholar]
  23. R Core Team R: A Language and Environment for Statistical Computing Vienna, Austria: R Foundation for Statistical Computing; 2015
    [Google Scholar]
  24. Lawrence M, Huber W, Pagès H, Aboyoun P, Carlson M et al. Software for computing and annotating genomic ranges. PLoS Comput Biol 2013;9:e1003118 [CrossRef][PubMed]
    [Google Scholar]
  25. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550 [CrossRef][PubMed]
    [Google Scholar]
  26. Liu G, Casqueiro J, Bañuelos O, Cardoza RE, Gutiérrez S et al. Targeted inactivation of the mecB gene, encoding cystathionine-γ-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes. J Bacteriol 2001;183:1765–1772 [CrossRef][PubMed]
    [Google Scholar]
  27. Terfehr D, Dahlmann TA, Specht T, Zadra I, Kürnsteiner H et al. Genome sequence and annotation of Acremonium chrysogenum, producer of the β-lactam antibiotic cephalosporin C. Genome Announc 2014;2:e00948-14 [CrossRef][PubMed]
    [Google Scholar]
  28. Topczewski J, Sienko M, Paszewski A. Cloning and characterization of the Aspergillus nidulans cysB gene encoding cysteine synthase. Curr Genet 1997;31:348–356 [CrossRef][PubMed]
    [Google Scholar]
  29. Sieńko M, Topczewski J, Paszewski A. Structure and regulation of cysD, the homocysteine synthase gene of Aspergillus nidulans. Curr Genet 1998;33:136–144[PubMed][CrossRef]
    [Google Scholar]
  30. Thomas D, Surdin-Kerjan Y. Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 1997;61:503–532[PubMed]
    [Google Scholar]
  31. Sohn MJ, Yoo SJ, Oh DB, Kwon O, Lee SY et al. Novel cysteine-centered sulfur metabolic pathway in the thermotolerant methylotrophic yeast Hansenula polymorpha. PLoS One 2014;9:e100725 [CrossRef][PubMed]
    [Google Scholar]
  32. Brzywczy J, Paszewski A. Sulfur amino acid metabolism in Schizosaccharomyces pombe: occurrence of two O-acetylhomoserine sulfhydrylases and the lack of the reverse transsulfuration pathway. FEMS Microbiol Lett 1994;121:171–174 [CrossRef][PubMed]
    [Google Scholar]
  33. Brzywczy J, Sieńko M, Kucharska A, Paszewski A. Sulphur amino acid synthesis in Schizosaccharomyces pombe represents a specific variant of sulphur metabolism in fungi. Yeast 2002;19:29–35 [CrossRef][PubMed]
    [Google Scholar]
  34. Paszewski A, Grabski J. Regulation of S-amino acids biosynthesis in Aspergillus nidulans. Mol Gen Genet 1974;132:307–320 [CrossRef]
    [Google Scholar]
  35. Paszewski A, Grabski J. Enzymatic lesions in methionine mutants of Aspergillus nidulans: role and regulation of an alternative pathway for cysteine and methionine synthesis. J Bacteriol 1975;124:893–904[PubMed]
    [Google Scholar]
  36. Kerr DS. O-Acetylhomoserine sulfhydrylase from Neurospora. Purification and consideration of its function in homocysteine and methionine synthesis. J Biol Chem 1971;246:95–102[PubMed]
    [Google Scholar]
  37. Pieniazek NJ, Bal J, Balbin E, Stepién PP. An Aspergillus nidulans mutant lacking serine transacetylase: evidence for two pathways of cysteine biosynthesis. Mol Gen Genet 1974;132:363–366 [CrossRef][PubMed]
    [Google Scholar]
  38. Brzywczy J, Natorff R, Sieńko M, Paszewski A. Multiple fungal enzymes possess cysteine synthase activity in vitro. Res Microbiol 2007;158:428–436 [CrossRef][PubMed]
    [Google Scholar]
  39. Tollnick C, Seidel G, Beyer M, Schügerl K. Investigations of the production of cephalosporin C by Acremonium chrysogenum. Adv Biochem Eng Biotechnol 2004;86:1–45[PubMed]
    [Google Scholar]
  40. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine, 5th ed. Oxford: Oxford University Press; 2015;[CrossRef]
    [Google Scholar]
  41. Penninckx MJ, Elskens MT. Metabolism and functions of glutathione in micro-organisms. Adv Microb Physiol 1993;34:239–301[PubMed][CrossRef]
    [Google Scholar]
  42. Fahey RC, Newton GL, Arrick B, Overdank-Bogart T, Aley SB. Entamoeba histolytica: a eukaryote without glutathione metabolism. Science 1984;224:70–72 [CrossRef][PubMed]
    [Google Scholar]
  43. Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983;52:711–760 [CrossRef][PubMed]
    [Google Scholar]
  44. Elskens MT, Jaspers CJ, Penninckx MJ. Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae. J Gen Microbiol 1991;137:637–644 [CrossRef][PubMed]
    [Google Scholar]
  45. Emri T, Pócsi I, Szentirmai A. Changes in the glutathione (GSH) metabolism of Penicillium chrysogenum grown on different nitrogen, sulphur and carbon sources. J Basic Microbiol 1998;38:3–8 [CrossRef]
    [Google Scholar]
  46. Paietta JV. Regulation of sulfur metabolism in mycelial fungi. In Brambl R, Marzluf GA. (editors) In Biochemistry and Molecular Biology Berlin: Springer Berlin Heidelberg; 2004; pp.369–383[CrossRef]
    [Google Scholar]
  47. Sieńko M, Natorff R, Skoneczny M, Kruszewska J, Paszewski A et al. Regulatory mutations affecting sulfur metabolism induce environmental stress response in Aspergillus nidulans. Fungal Genet Biol 2014;65:37–47 [CrossRef][PubMed]
    [Google Scholar]
  48. Sieńko M, Natorff R, Owczarek S, Olewiecki I, Paszewski A. Aspergillus nidulans genes encoding reverse transsulfuration enzymes belong to homocysteine regulon. Curr Genet 2009;55:561–570 [CrossRef][PubMed]
    [Google Scholar]
  49. Wang H, Pan Y, Hu P, Zhu Y, Li J et al. The autophagy-related gene Acatg1 is involved in conidiation and cephalosporin production in Acremonium chrysogenum. Fungal Genet Biol 2014;69:65–74 [CrossRef][PubMed]
    [Google Scholar]
  50. Bartoshevich YuE, Zaslavskaya PL, Novak MJ, Yudina OD. Acremonium chrysogenum differentiation and biosynthesis of cephalosporin. J Basic Microbiol 1990;30:313–320 [CrossRef][PubMed]
    [Google Scholar]
  51. Nash CH, Huber FM. Antibiotic synthesis and morphological differentiation of Cephalosporium acremonium. Appl Microbiol 1971;22:6–10[PubMed]
    [Google Scholar]
  52. Drew SW, Winstanley DJ, Demain AL. Effect of norleucine on mycelial fragmentation in Cephalosporium acremonium. Appl Environ Microbiol 1976;31:143–145[PubMed]
    [Google Scholar]
  53. Matsumura M, Imanaka T, Yoshida T, Taguchi H. Effect of glucose and methionine consumption rates on cephalosporin C production by Cephalosporium acremonium. J Ferment Technol 1978;56:345–353
    [Google Scholar]
  54. Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene 1992;110:119–122 [CrossRef][PubMed]
    [Google Scholar]
  55. Nowrousian M, Cebula P. The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora. BMC Microbiol 2005;5:1–10 [CrossRef]
    [Google Scholar]
  56. Kück U, Hoff B. Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi. Fungal Genet Rep 2006;53:9–11 [CrossRef]
    [Google Scholar]
  57. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–3402 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000474
Loading
/content/journal/micro/10.1099/mic.0.000474
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error