title: Metabolic engineering of the fungal D-galacturonate pathway for L-ascorbic acid production.
abstract: BACKGROUND
  Synthetic L-ascorbic acid (vitamin C) is widely used as a preservative and nutrient in food and pharmaceutical industries. In the current production method, D-glucose is converted to L-ascorbic acid via several biochemical and chemical steps. The main source of L-ascorbic acid in human nutrition is plants. Several alternative metabolic pathways for L-ascorbic acid biosynthesis are known in plants. In one of them, D-galacturonic acid is the precursor. D-Galacturonic acid is also the main monomer in pectin, a plant cell wall polysaccharide. Pectin is abundant in biomass and is readily available from several waste streams from fruit and sugar processing industries.
  
  
  RESULTS
  In the present work, we engineered the filamentous fungus Aspergillus niger for the conversion of D-galacturonic acid to L-ascorbic acid. In the generated pathway, the native D-galacturonate reductase activity was utilized while the gene coding for the second enzyme in the fungal D-galacturonic acid pathway, an L-galactonate consuming dehydratase, was deleted. Two heterologous genes coding for enzymes from the plant L-ascorbic acid pathway--L-galactono-1,4-lactone lactonase from Euglena gracilis (EgALase) and L-galactono-1,4-lactone dehydrogenase from Malpighia glabra (MgGALDH)--were introduced into the A. niger strain. Alternatively, an unspecific L-gulono-1,4-lactone lactonase (smp30) from the animal L-ascorbic acid pathway was introduced in the fungal strain instead of the plant L-galactono-1,4-lactone lactonase. In addition, a strain with the production pathway inducible with D-galacturonic acid was generated by using a bidirectional and D-galacturonic acid inducible promoter from the fungus. Even though, the lactonase enzyme activity was not observed in the resulting strains, they were capable of producing L-ascorbic acid from pure D-galacturonic acid or pectin-rich biomass in a consolidated bioprocess. Product titers up to 170 mg/l were achieved.
  
  
  CONCLUSIONS
  In the current study, an L-ascorbic acid pathway using D-galacturonic acid as a precursor was introduced to a microorganism for the first time. This is also the first report on an engineered filamentous fungus for L-ascorbic acid production and a proof-of-concept of consolidated bioprocess for the production.
authors.affiliation: VTT Technical Research Centre of Finland, PO Box 1000, 02044 VTT, Espoo, Finland. joosu.kuivanen@vtt.fi.
authors.forename: Joosu
authors.initials: J
authors.lastname: Kuivanen
authors.affiliation: VTT Technical Research Centre of Finland, PO Box 1000, 02044 VTT, Espoo, Finland. merja.penttila@vtt.fi.
authors.forename: Merja
authors.initials: M
authors.lastname: Penttilä
authors.affiliation: VTT Technical Research Centre of Finland, PO Box 1000, 02044 VTT, Espoo, Finland. peter.richard@vtt.fi.
authors.forename: Peter
authors.initials: P
authors.lastname: Richard
mesh_terms: D001205:Ascorbic Acid / Q000096:biosynthesis*; D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D005056:Euglena gracilis / Q000201:enzymology; D005656:Fungal Proteins / Q000235:genetics; D006603:Hexuronic Acids / Q000378:metabolism*; D006836:Hydro-Lyases / Q000235:genetics; D029581:Malpighiaceae / Q000201:enzymology; D060847:Metabolic Engineering; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D006603:Hexuronic Acids; D010940:Plant Proteins; D015800:Protozoan Proteins; C007819:galacturonic acid; D044925:Oxidoreductases Acting on CH-CH Group Donors; C093910:galactonolactone dehydrogenase; D006836:Hydro-Lyases; D001205:Ascorbic Acid