Single Amino Acids as Sole Nitrogen Source for the Production of Lipids and Coenzyme Q by Thraustochytrium sp. RT2316-16.

This work analyzes the production of total lipids and the content of CoQ9 and CoQ10 in the biomass of Thraustochytrium sp. RT2316-16 grown in media containing a single amino acid at a concentration of 1 g L-1 as the sole nitrogen source; glucose (5 g L-1) was used as the carbon source. Biomass concentration and the content of total lipids and CoQ were determined as a function of the incubation time; ten amino acids were evaluated. The final concentration of the total biomass was found to be between 2.2 ± 0.1 (aspartate) and 3.9 ± 0.1 g L-1 (glutamate). The biomass grown in media containing glutamate, serine or phenylalanine reached a content of total lipids higher than 20% of the cell dry weight (DW) after 72, 60 and 72 h of incubation, respectively. The highest contents of CoQ9 (39.0 ± 0.7 µg g-1 DW) and CoQ10 (167.4 ± 3.4 mg g-1 DW) in the biomass of the thraustochytrid were obtained when glutamate and cysteine were used as the nitrogen source, respectively. Fatty acid oxidation, which decreased the total lipid content during the first 12 h of incubation, and the oxidation of hydrogen sulfide when cysteine was the nitrogen source, might be related to the content of CoQ10 in the biomass of the thraustochytrid.

Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism.

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.

Coenzyme Q in Thraustochytrium sp. RT2316-16: Effect of the Medium Composition.

Coenzyme Q (CoQ; ubiquinone) is an essential component of the respiratory chain. It is also a potent antioxidant that prevents oxidative damage to DNA, biological membranes, and lipoproteins. CoQ comprises a six-carbon ring with polar substituents that interact with electron acceptors and donors, and a hydrophobic polyisoprenoid chain that allows for its localization in cellular membranes. Human CoQ has 10 isoprenoid units (CoQ10) within the polyisoprenoid chain. Few microorganisms produce CoQ10. This work shows that Thraustochytrium sp. RT2316-16 produces CoQ10 and CoQ9. The CoQ10 content in RT2316-16 depended strongly on the composition of the growth medium and the age of the culture, whereas the CoQ9 content was less variable probably because it served a different function in the cell. Adding p-hydroxybenzoic acid to the culture media positively influenced the CoQ10 content of the cell. The absence of some B vitamins and p-aminobenzoic acid in the culture medium negatively affected the growth of RT2316-16, but reduced the decline in CoQ10 that otherwise occurred during growth. The highest content of CoQ9 and CoQ10 in the biomass were 855 µg g-1 and 10 mg g-1, respectively. The results presented here suggest that the thraustochytrid RT2316-16 can be a potential vehicle for producing CoQ10. Metabolic signals that trigger the synthesis of CoQ10 in RT2316-16 need to be determined for optimizing culture conditions.

Production of Carotenoids and Phospholipids by Thraustochytrium sp. in Batch and Repeated-Batch Culture.

The carotenogenic thraustochytrid Thraustochytrium sp. RT2316-16 was grown in batch and repeated-batch cultures using different feeds containing glucose, or glycerol, and yeast extract, for the production of lipids, phospholipids and carotenoids. RT2316-16 produced canthaxanthin, astaxanthin and ß-carotene. The effects of biotin, ascorbic acid, light and temperature were evaluated in some of the experiments. In 2-day-old batch cultures, the combined mass percentage of eicosapentaenoic acid and docosahexaenoic acid in total lipids was between 16.5% (glycerol-based medium in the dark; biomass concentration = 4.2 ± 1.1 g L-1) and 42.6% (glucose-based medium under light; biomass concentration = 3.3 ± 0.1 g L-1), decreasing to 3.8% and 6.1%, respectively, after day 4. In repeated-batch cultures, the total lipids in the biomass increased after glucose or glycerol was fed alone, whereas the total carotenoids (168 ± 7 µg g-1 dry weight (DW)) and phospholipids in the biomass increased after feeding with yeast extract. The biomass with the highest content of phospholipids (28.7 ± 4.3 mg g-1 DW) was obtained using a feed medium formulated with glycerol, yeast extract and ascorbic acid. Glycerol was the best carbon source for the production of a biomass enriched with total lipids (467 ± 45 mg g-1 DW). The composition of carotenoids depended strongly on the composition of the feed. Repeated-batch cultures fed with yeast extract contained canthaxanthin as the main carotenoid, whereas in the cultures fed only with glucose, the biomass contained mainly ß-carotene.

Nitrogen Sources Affect the Long-Chain Polyunsaturated Fatty Acids Content in Thraustochytrium sp. RT2316-16.

The psychrophilic marine microorganism Thraustochytrium sp. RT2316-16 can produce carotenoids as well as lipids containing the omega-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid and docosahexaenoic acid. This work reports on the effects of the composition of the culture medium, including certain amino acids, on growth and lipid synthesis by RT2316-16. Compared with the culture on glutamate, the use of lysine, alanine, or serine, increased the content of the omega-3 PUFA in total lipids. In the media that contained yeast extract, glutamate, and glucose, lipid accumulation occurred when organic ammonium was exhausted earlier than glucose. In contrast, lipid mobilization was promoted if glucose was exhausted while organic ammonium (supplied by yeast extract and glutamate) remained in the medium. The total content of carotenoids in the lipid-free biomass decreased during the first 12 to 24 h of culture, simultaneously with a decrease in the total lipid content of the biomass. The experimental data suggested a possible interrelationship between the metabolism of carotenoids and lipids. A high content of omega-3 PUFA in the total lipids could be obtained by growing the thraustochytrid in a medium with a low glucose concentration (6 g L-1) and a high concentration of organic nitrogen (yeast extract 12 g L-1; glutamate 1.06 g L-1), after glucose was exhausted. These observations may guide the development of a strategy to enhance omega-3 PUFA in the biomass.