The content of (a) ALA, (b) EPA, (c) DHA, (d) α-tocopherol and (e) phytol in <i>E</i>. <i>gracilis</i> biomass from autotrophic (23 °C square, 30 °C triangle), heterotrophic (23 °C solid circle) and mixotrophic (23 °C circle with cross) cultivations.

<p>The content of (a) ALA, (b) EPA, (c) DHA, (d) α-tocopherol and (e) phytol in <i>E</i>. <i>gracilis</i> biomass from autotrophic (23 °C square, 30 °C triangle), heterotrophic (23 °C solid circle) and mixotrophic (23 °C circle with cross) cultivations.</p

Specific growth rates (μ) during exponential phase of mixotrophic (primarily phototrophic growth) and heterotrophic cultures of <i>E</i>. <i>gracilis</i> at 23 and 27 °C.

<p>Specific growth rates (μ) during exponential phase of mixotrophic (primarily phototrophic growth) and heterotrophic cultures of <i>E</i>. <i>gracilis</i> at 23 and 27 °C.</p

Growth of photoautotrophic cultures of <i>E</i>. <i>gracilis</i> at 23 (circle) or 30 °C (upward and downward triangles) with two (square) or three (circle and triangles) light sources of 400 μE m^-2 s^-1 intensity (2 x 400 or 3 x 400 μE m^-2 s^-1).

<p>(a), Growth curves measured by cell dry weight. Error bars (which were generally smaller than the symbol) represent the standard error of the mean (n = 2–6). (b), Natural logarithms (ln) of cell density measured by OD₇₈₀, and the least square regression lines.</p

The content (as percent, a and c) and concentration (b and d) of protein (a and b) and paramylon (c and d) in photoautotrophically grown <i>E</i>. <i>gracilis</i>.

<p>Cultures grown at 23 °C (square) and at 30 °C (up and down triangles) with 3 x 400 μmol m^-2 s^-1 light. One culture at 30 °C provided samples during the initial 6 days of growth, whereas the second culture included only an end-point sample, the data from which is included here for comparison with the longer cultivation at 23 °C.</p

Total lipid (a) content, as percentage of dry weight, and (b) concentration of <i>E</i>. <i>gracilis</i> biomass generated in autotrophic conditions.

<p>Cultures grown at 23 °C (circle and square) with 2 x 400 (circle) or 3 x 400 μmol m^-2 s^-1 (square) light or at 30 °C (triangle) with 3 x 400 μmol m^-2 s^-1 light.</p

Cultivation conditions and analyses carried out from each cultivation.

<p>Cultivation conditions and analyses carried out from each cultivation.</p

Specific growth rates (μ) and volumetric growth rates (r-lin) of photoautotrophic cultures of <i>E</i>. <i>gracilis</i> at 23 and 30 °C with 2 x 400 or 3 x 400 μmol m^-2 s^-1 light.

<p>Specific growth rates (μ) and volumetric growth rates (r-lin) of photoautotrophic cultures of <i>E</i>. <i>gracilis</i> at 23 and 30 °C with 2 x 400 or 3 x 400 μmol m^-2 s^-1 light.</p

Table_1_Enhanced Triacylglycerol Production With Genetically Modified Trichosporon oleaginosus.PDF

<p>Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21–0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.</p