초록
<P><B>Background</B></P><P>L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this “L” enantiomer, we engineered <I>Kluyveromyces lactis</I> with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from <I>Arabidopsis thaliana</I>.</P><P><B>Results</B></P><P>Plasmids were constructed and modified such that the cloned plant genes were targeted to the <I>K. lactis</I> LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon <I>K. lactis</I> transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the <I>S. cerevisiae</I> promoters <I>GPD1</I> and <I>ADH1</I> respectively. The expression in <I>K. lactis,</I> of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30 mg.L<SUP>-1</SUP> of L-ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose.</P><P><B>Conclusions</B></P><P>This work is the first attempt to engineer <I>K. lactis</I> cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of “L” isomers precursors in the culture medium. We have engineered <I>K. lactis</I> strains capable of converting lactose and D-galactose into L-galactose, by the integration of the genes from the <I>A. thaliana</I> L-galactose pathway. L-galactose is a rare sugar, which is one of the main precursors for L-AA production.</P>