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Journal of Experimental Botany, Vol. 54, No. 389, pp. 1841-1849, August 1, 2003
© 2003 Oxford University Press

The pathway of L-ascorbic acid biosynthesis in the colourless microalga Prototheca moriformis

Received 11 February 2003; Accepted 2 May 2003

Jeffrey Allan Running*,, Richard Paul Burlingame{dagger}, and Alan Berry{ddagger},

Bio-Technical Resources, 1035 South 7th Street, Manitowoc, WI 54220, USA

* To whom correspondence should be sent. Fax: +1 920 684 5519. E-mail:jeff{at}biotechresources.com
{dagger} Present address: Dyadic International Inc., 140 Intracoastal Pointe Drive, Suite 404, Jupiter, FL 33477-5094, USA.
{ddagger} Present address: Biotechnology Department, Research and Development, Roche Vitamins AG, CH-4070 Basel, Switzerland.
Abbreviations: AA, L-ascorbic acid; GMP, GDP-Mannose pyrophosphorylase; L-GalL, L-galactono-1,4-lactone; L-GalDH, L-galactose dehydrogenase; PMI, phosphomannose isomerase; PMM, phosphomannomutase.

When mutant strain UV77–247 of Prototheca moriformis Kruger was fed D-[1-13C]Glc, it synthesized L-ascorbic acid (AA) with approximately three-quarters of the label at the C-1 position and the remaining label at the C-6 position, showing that AA is made by a non-inversion (retention) pathway, i.e. C-1 of Glc becomes C-1 of AA. The label present at C-6 is consistent with the glycolytic conversion of Glc to 3-carbon intermediates and subsequent gluconeogenesis. Compounds suggested as intermediates in inversion-type pathways were not converted to AA. Most strains converted Man to AA at a rate greater than they did Glc. Enzyme activities leading from Fru-6-P to the formation of GDP-Man were identified in all strains, but none of these activities correlated with the mutants’ abilities to accumulate AA. However, there was a strong correlation between GDP-Man-3,5-epimerase activity and AA accumulation. Wild-type P. moriformis ATCC 75669 and mutant strains of varying AA-synthesizing abilities rapidly converted L-Gal or L-galactono-1,4-lactone to AA. Based on this data, a biosynthetic pathway from Glc to AA is proposed in which the epimerase is the rate-limiting activity in AA synthesis.

Key words: GDP-Mannose-3,5-epimerase, L-ascorbic acid, microalga, Prototheca.


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