Growth, fatty acid profile in major lipid classes and lipid fluidity of Aurantiochytrium mangrovei SK-02 As a function of growth temperature.

Aurantiochytrium mangrovei Sk-02 was grown in a medium containing glucose (40 g/l), yeast extract (10 g/L) and sea salts (15 g/L) at temperatures ranging from 12 to 35°C. The fastest growth (µmax= 0.15 h(-1)) and highest fatty acid content of 415 mg/g-dry cell weight were found in the cells grown at 30°C. However, the cells grown at 12°C showed the highest percentage of polyunsaturated fatty acid (PUFA) (48.6% of total fatty acid). The percentage of docosahexaenoic acid (DHA) and pentadecanoic acid (C15:0) decreased with an increase in the growth temperature, whereas, palmitic acid (C16:0), stearic acid (C18:0) and DPA (C22:5n6) increased with an increase in the growth temperature. The composition of the major lipid class (%w/w) was slightly affected by the growth temperature. The fluidity of the organelle membrane or intracellular lipid (by DPH measurement) decreased with an increase in the growth temperatures, while the plasma membrane fluidity (by TMA-DPH measurement) could still maintain its fluidity in a wide range of temperatures (15 - 37°C). Furthermore, the distribution of DHA was found to be higher (36 - 54%) in phospholipid (PL) as compared to neutral lipid (NL) (20 - 41%).

Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation.

Baffled shake flask cultivation of Aurantiochytrium sp. B-072 was carried out at in a glucose-monosodium glutamate mineral medium at different C/N-ratios (30-165) with glucose fixed at 90 g/L. With increasing C/N-ratio, a modest increase in lipid content (60 to 73 % w/w) was observed whereas fat-free biomass decreased but overall biomass showed little variation. FA-profiles were not affected to a large extent by C/N-ratio and absolute docosahexaenoic (DHA)-levels fell in narrow range (5-6 g/L). However at C/N > 64 a rapid decrease in lipid synthetic rate and/or incomplete glucose utilization occurred. Glucose and FA-fluxes based on fat-free biomass peaked at a C/N ratio of 56. This condition was chosen for calculation of the redox balance (NAD(P)H) and energy (ATP) requirement and to estimate the in vivo P/O ratio during the main period of fatty acid biosynthesis. Several models with different routes for NADPH, acetyl-CoA formation and re-oxidation of OAA formed via ATP-citrate lyase were considered as these influence the redox- and energy balance. As an example, using a commonly shown scheme whereby NADPH is supplied by a cytosolic "transhydrogenase cycle" (pyruvate-OAA-malate-pyruvate) and OAA formed by ATP-citrate lyase is recycled via import into the mitochondria as malate, the calculated NADPH-requirement amounted to 5.5 with an ATP-demand of 10.5 mmol/(g fat-free biomass x h) and an in vivo P/O-ratio (not including non-growth associated maintenance) of 1.6. The lowest ATP requirement is found when acetyl-CoA would be transported directly from the mitochondria to the cytosol by carnitine acetyltransferase. Assay of some enzymes critical for NADPH supply indicates that activity of glucose-6-phosphate dehydrogenase, the first enzyme in the HMP pathway, is far insufficient for the required NADPH-flux and malic enzyme must be a major source. Activity of the latter (ca. 300 mU/mg protein) far exceeds that in oleaginous fungi and yeast.

Coconut water as a medium additive for the production of docosahexaenoic acid (C22:6 n3) by Schizochytrium mangrovei Sk-02.

The effect of coconut water (CW) on biomass and docosahexaenoic acid (DHA, C22:6 n3) formation by Schizochytrium mangrovei Sk-02 was studied in a yeast extract-diluted sea water medium. Optimal CW-level was ca. 33% (v/v), resulting in a biomass level of 28 g/l with a DHA-content of 20% (w/w) or 6 g DHA/l, almost 50% higher than in non-supplemented cultures at the same initial sugar level. Study on the growth-promoting effects of coconut water suggested that it could be (partially) mimicked by addition of trace elements; the fatty acids present in CW did not appear to be incorporated or effect fatty acid formation by the organism. CW-addition was also effective in media with other nitrogen sources such as casitone, peptone and tryptone. Its inclusion (at 50% v/v) increased biomass levels two-to-three-fold with concomitant increases in the DHA-level.

Improved docosahexaenoic acid production in Aurantiochytrium by glucose limited pH-auxostat fed-batch cultivation.

Fed-batch, pH auxostat cultivation of the docosahexaenoic acid (DHA)-producing microorganism Aurantiochytrium B072 was performed to obtain high cell density and record high productivity of both total fatty acid (TFA) and DHA. Using glucose feeding by carbon excess (C-excess) and by C-limitation at various feeding rates (70%, 50% or 20% of C-excess), high biomass density was obtained and DHA/TFA content (w/w) was improved from 30% to 37% with a 50% glucose feed rate when compared with C-excess. To understand the biochemistry behind these improvements, lipogenic enzyme assays and in silico metabolic flux calculations were used and revealed that enzyme activity and C-fluxes to TFA were reduced with C-limited feeding but that the carbon flux to the polyketide synthase pathway increased relative to the fatty acid synthase pathway. As a result, a new strategy to improve the DHA to TFA content while maintaining relatively high DHA productivity is proposed.

Growth, fatty acid profile in major lipid classes and lipid fluidity of Aurantiochytrium mangrovei Sk-02 as a function of growth temperature

Aurantiochytrium mangrovei Sk-02 was grown in a medium containing glucose (40 g/l), yeast extract (10 g/L) and sea salts (15 g/L) at temperatures ranging from 12 to 35°C. The fastest growth (µmax= 0.15 h-1) and highest fatty acid content of 415 mg/g-dry cell weight were found in the cells grown at 30°C. However, the cells grown at 12°C showed the highest percentage of polyunsaturated fatty acid (PUFA) (48.6% of total fatty acid). The percentage of docosahexaenoic acid (DHA) and pentadecanoic acid (C15:0) decreased with an increase in the growth temperature, whereas, palmitic acid (C16:0), stearic acid (C18:0) and DPA (C22:5n6) increased with an increase in the growth temperature. The composition of the major lipid class (%w/w) was slightly affected by the growth temperature. The fluidity of the organelle membrane or intracellular lipid (by DPH measurement) decreased with an increase in the growth temperatures, while the plasma membrane fluidity (by TMA-DPH measurement) could still maintain its fluidity in a wide range of temperatures (15 - 37°C). Furthermore, the distribution of DHA was found to be higher (36 - 54%) in phospholipid (PL) as compared to neutral lipid (NL) (20 - 41%).

Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation

Baffled shake flask cultivation of Aurantiochytrium sp. B-072 was carried out at in a glucose-monosodium glutamate mineral medium at different C/N-ratios (30-165) with glucose fixed at 90 g/L. With increasing C/N-ratio, a modest increase in lipid content (60 to 73 % w/w) was observed whereas fat-free biomass decreased but overall biomass showed little variation. FA-profiles were not affected to a large extent by C/N-ratio and absolute docosahexaenoic (DHA)-levels fell in narrow range (5-6 g/L). However at C/N > 64 a rapid decrease in lipid synthetic rate and/or incomplete glucose utilization occurred. Glucose and FA-fluxes based on fat-free biomass peaked at a C/N ratio of 56. This condition was chosen for calculation of the redox balance (NAD(P)H) and energy (ATP) requirement and to estimate the in vivo P/O ratio during the main period of fatty acid biosynthesis. Several models with different routes for NADPH, acetyl-CoA formation and re-oxidation of OAA formed via ATP-citrate lyase were considered as these influence the redox- and energy balance. As an example, using a commonly shown scheme whereby NADPH is supplied by a cytosolic "transhydrogenase cycle" (pyruvate-OAA-malate-pyruvate) and OAA formed by ATP-citrate lyase is recycled via import into the mitochondria as malate, the calculated NADPH-requirement amounted to 5.5 with an ATP-demand of 10.5 mmol/(g fat-free biomass x h) and an in vivo P/O-ratio (not including non-growth associated maintenance) of 1.6. The lowest ATP requirement is found when acetyl-CoA would be transported directly from the mitochondria to the cytosol by carnitine acetyltransferase. Assay of some enzymes critical for NADPH supply indicates that activity of glucose-6-phosphate dehydrogenase, the first enzyme in the HMP pathway, is far insufficient for the required NADPH-flux and malic enzyme must be a major source. Activity of the latter (ca. 300 mU/mg protein) far exceeds that in oleaginous fungi and yeast.