Characterisation of Chlorella pyrenoidosa L-ascorbic acidaccumulating mutants: Identification of an enhanced biosynthetic enzyme activity and cloning of the putative gene from Arabidopsis thaliana

We have undertaken a comparative study of L-ascorbic acid (L-AA) biosynthesis in wild-type Chlorella pyrenoidosa and two mutant strains containing enhanced LAA content. Both mutant strains synthesized L-AA more efficiently from the distant precursors D-glucose or Dmannose whilst enhanced biosynthesis was not observed from the immediate precursors L-galactose or L-galactono- 1,4-lactone. In vitro assay of individual biosynthetic steps revealed that only one enzyme exhibited enhanced activity in both mutant strains, a putative GDP-L-galactose pyrophosphatase. Both mutant strains were found to contain higher concentrations of free Lgalactose than the wild-type strain suggesting that GDPL- galactose pyrophophatase andyor L-galactose-1- phosphate phosphatase activity were also enhanced in vivo. In order to simplify cloning of the pyrophosphatase gene, we chose to purify the enzyme from Arabidopsis thaliana due to the availability of the genome sequence. The activity was partially purified and a number of proteins were N-terminal sequenced by Edman degradation, one of which corresponded to a putative nucleotide pyrophosphatase- like protein. Nucleotide pyrophosphatase protein concentrations correlated well with enzyme activity as judged by Coomassie staining on polyacrylamide gels. Current efforts are focused on in vitro characterization of the recombinant enzyme and in vivo analysis of protein function.