l-Ascorbic Acid Biosynthesis in Ochromonas danica

Abstract

Ochromoaas danica Pringsheim, a freshwater chrysomonad, converts D-glucose into L-ascorbkc acid over a metabolic pathway that 'inverts' the carbon chain of the sugar. In this respect, L-ascorbic acid formation resembles that found in ascorbic acid-synthesizing animals. It differs from this process in that D-galacturonate and L-galactono-1,4-Iactone, rather than D-gucuronate and L-guloo-1,4-lactone, enhance production of ascorbic acid and repress the incorporation of 14C from D-l1-14ClgIucose into ascorbic acid.Two mutually exclusive, biosynthetic pathways of AA3 formation are found in eukaryotic organisms. In both, D-glucose is implicated as the six-carbon precursor on the basis of studies with radioactive specifically labeled sugars. Both pathways conserve the six-carbon chain. One 'inverts' the sequence of carbons, as referred to the parent sugar by reduction of C-1 and oxidation of C-5 (or C4) and C-6 with conservation of the chirality of C-2 and C-3. AA-Synthesizing animals such as the rat utilize this process according to the following scheme:lactone -. L-Ascorbate. The other pathway requires that C-I and C-2 (or C-3) be oxidized while an epimerization reverses the configuration of C-5. Higher plants utilize this latter process, which conserves the hydroxymethyl function of C-6 of hexose (15) 4-lactone (4, 6, 9, 19). Inasmuch as radiolabeling studies exclude these compounds as intermediates in the overall pathway from D-glucose to AA, these synthetic events appear to be dependent upon the exogenous source or, at best, salvage mechanisms (7 and L-galactono-1,4-lactone were intermediates. D-Glucurono-3,6-lactone and L-gulono-1,4-lactone also effected AA synthesis, and these compounds were included in their scheme as a minor alternate route that differed from the animal pathway in one respect, reduction of D-glucurono-3,6-lactone to L-gulono-1,4-lactone rather than reduction of D-glucuronate to L-gulonate (23).Ochromonas danica Pringsheim, a unicellular chrysophycean alga with phagotrophic, heterotrophic, photoheterotrophic, and photoautotrophic properties, provides an exceptional opportunity to explore the phylogenetic connection between animal and plant as it pertains to AA biosynthesis. 0. danica synthesizes and secretes into the medium a number of vitamins, including AA, when grown in an artificial medium (2). Advantage has been taken of this observation to explore the conversion of specifically labeled D-glucose into AA and to test the effect of possible intermediates in this process. MATERIALS AND METHODSChemicals. L-[l-14C]Ascorbic acid and specifically labeled forms of D-glucose were purchased from New England Nuclear Corp. Chemicals, including those used to prepare culture media, were reagent grade.Algal Culture. 0. danica Pringsheim was obtained from the Culture Collection of Algae at the University of Texas at Austin. Cells were dark-grown at 25°C in chemically defined medium containing 1% D-glucose (1). Inoculum (10 ml, approx. 108 cells) was added to 100 ml of medium. After 14 d, the enti...

Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase

“…Unicellular green algae such as the chlorophytes Chlorella pyrenoidosa and Prototheca moriformis can synthesize L-ascorbate using the L-galactose pathway (10 -12). Two other photosynthetic unicellular protists (Euglena gracilis and Ochromonas danica) (13,14) and a diatom (Cyclotella cryptica) utilize the inversion pathway commonly found in animals (supplemental Fig. S1) (15).…”

Effect of Lignocellulose Related Compounds on Microalgae Growth and Product Biosynthesis: A Review

“…Addition of 2.5 g/L glucuronic acid failed to enhance ascorbic acid synthesis [89]. However, mixotrophic cultivation of microalga Euglena gracilis caused 2-fold and 4-fold increase in ascorbic acid production with 2.5 g/L glucuronic and galacturonic acid, respectively, as a comparison to the control grown in the presence of light, but without any added sugars or sugar acids.…”

“…Addition of 2.5 g/L galacturonic acid to the microalga Ochromonas danica cultivated on 1 g/L glucose in the presence of light resulted in 3.3 fold increase in ascorbic acid production when compared to experiments, where Ochromonas was cultivated mixotrophically on glucose. Addition of 2.5 g/L glucuronic acid failed to enhance ascorbic acid synthesis [89]. However, mixotrophic cultivation of microalga Euglena gracilis caused 2-fold and 4-fold increase in ascorbic acid production with 2.5 g/L glucuronic and galacturonic acid, respectively, as a comparison to the control grown in the presence of light, but without any added sugars or sugar acids.…”

Uptake of l-Ascorbate by Intact Spinach Chloroplasts

“…Considerable amounts of L-ascorbic acid are found in algae (10,17,33) and in fruits, storage organs, and leaves of higher plants (26). Inasmuch as 30 to 40% of the ascorbate content of spinach leaf protoplasts was found to be associated with the chloroplast fraction (8), a considerable portion of the ascorbate of green leaves appears to be localized within the chloroplasts.…”

“…Although the biosynthesis of L-ascorbic acid in plants has not yet been completely elucidated, it is well established that it originates from D-glucose. In contrast to the biosynthetic route in animals or algae (17), ascorbate biosynthesis in higher plants proceeds via oxidation at C-1, epimerization at C-5, and a second oxidation at C-2 or C-3 (12,16; for earlier references, see 9). The enzymes catalyzing this reaction sequence have not yet been localized in the cell.…”