Nine strains of Chlorella protothecoides and 43 strains representing the five species of Prototheca were screened in flask culture for their ability to synthesize L-ascorbic acid (AA). Ascorbic acid was detected in all strains, ranging from 4.8 to 0.38 mg AA g x (-1) of dry cells. Organisms selected for further study grew well and maintained their AA productivity above a pH of 3.5. They can produce AA using a variety of carbon and nitrogen sources. Aerobic fermentation of selected strains resulted in extracellular accumulation of AA up to 76 mg x l(-1). By classical mutagenesis and selection methods, we created mutants of Prototheca moriformis ATCC 75669 that produced greater quantities of AA than the wild-type strain (78.4 vs 21.9 mg AA g x (-1) of cells). A process based on extracellular production could greatly reduce the cost of AA manufacture by eliminating the need for extraction of the AA from the cells.

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.

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