title: Synthesis of an antiviral drug precursor from chitin using a saprophyte as a whole-cell catalyst.
abstract: BACKGROUND
  Recent incidents, such as the SARS and influenza epidemics, have highlighted the need for readily available antiviral drugs. One important precursor currently used for the production of Relenza, an antiviral product from GlaxoSmithKline, is N-acetylneuraminic acid (NeuNAc). This substance has a considerably high market price despite efforts to develop cost-reducing (biotechnological) production processes. Hypocrea jecorina (Trichoderma reesei) is a saprophyte noted for its abundant secretion of hydrolytic enzymes and its potential to degrade chitin to its monomer N-acetylglucosamine (GlcNAc). Chitin is considered the second most abundant biomass available on earth and therefore an attractive raw material.
  
  
  RESULTS
  In this study, we introduced two enzymes from bacterial origin into Hypocrea, which convert GlcNAc into NeuNAc via N-acetylmannosamine. This enabled the fungus to produce NeuNAc from the cheap starting material chitin in liquid culture. Furthermore, we expressed the two recombinant enzymes as GST-fusion proteins and developed an enzyme assay for monitoring their enzymatic functionality. Finally, we demonstrated that Hypocrea does not metabolize NeuNAc and that no NeuNAc-uptake by the fungus occurs, which are important prerequisites for a potential production strategy.
  
  
  CONCLUSIONS
  This study is a proof of concept for the possibility to engineer in a filamentous fungus a bacterial enzyme cascade, which is fully functional. Furthermore, it provides the basis for the development of a process for NeuNAc production as well as a general prospective design for production processes that use saprophytes as whole-cell catalysts.
authors.affiliation: Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorfer Str. 1a, A-1060 Wien, Austria.
authors.forename: Matthias G
authors.initials: MG
authors.lastname: Steiger
authors.forename: Astrid R
authors.initials: AR
authors.lastname: Mach-Aigner
authors.forename: Rita
authors.initials: R
authors.lastname: Gorsche
authors.forename: Erwin E
authors.initials: EE
authors.lastname: Rosenberg
authors.forename: Marko D
authors.initials: MD
authors.lastname: Mihovilovic
authors.forename: Robert L
authors.initials: RL
authors.lastname: Mach
mesh_terms: D017033:Anabaena / Q000201:enzymology / Q000235:genetics; D000998:Antiviral Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D016123:Campylobacter jejuni / Q000201:enzymology* / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002686:Chitin / Q000378:metabolism*; D006595:Hexosamines / Q000378:metabolism; D035901:Hypocrea / Q000235:genetics* / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D000998:Antiviral Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D006595:Hexosamines; D002686:Chitin; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; C002022:N-acetylmannosamine