초록
<P><B>Background</B></P><P>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.</P><P><B>Results</B></P><P>In the present work, we engineered the filamentous fungus <I>Aspergillus niger</I> 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 <I>Euglena gracilis</I> (<I>EgALase</I>) and L-galactono-1,4-lactone dehydrogenase from <I>Malpighia glabra</I> (<I>MgGALDH</I>) – were introduced into the <I>A. niger</I> strain. Alternatively, an unspecific L-gulono-1,4-lactone lactonase (<I>smp30</I>) 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.</P><P><B>Conclusions</B></P><P>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.</P>