Cocktail biosynthesis of triacylglycerol by rational modulation of diacylglycerol acyltransferases in industrial oleaginous Aurantiochytrium

Background Triacylglycerol (TAG) is an important storage lipid in organisms, depending on the degree of unsaturation of fatty acid molecules attached to glycerol; it is usually used as the feedstock for nutrition or biodiesel. However, the mechanism of assembly of saturated fatty acids (SFAs) or polyunsaturated fatty acids (PUFAs) into TAGs remains unclear for industrial oleaginous microorganism. Results Diacylglycerol acyltransferase (DGAT) is a key enzyme for TAG synthesis. Hence, ex vivo (in yeast), and in vivo functions of four DGAT2s (DGAT2A, DGAT2B, DGAT2C, and DGAT2D) in industrial oleaginous thraustochytrid Aurantiochytrium sp. SD116 were analyzed. Results revealed that DGAT2C was mainly responsible for connecting PUFA to the sn-3 position of TAG molecules. However, DGAT2A and DGAT2D target SFA and/or MUFA. Conclusions There are two specific TAG assembly routes in Aurantiochytrium. The “saturated fatty acid (SFA) TAG lane” primarily produces SFA-TAGs mainly mediated by DGAT2D whose function is complemented by DGAT2A. And, the “polyunsaturated fatty acid (PUFA) TAG lane” primarily produces PUFA-TAGs via DGAT2C. In this study, we demonstrated the functional distribution pattern of four DGAT2s in oleaginous thraustochytrid Aurantiochytrium, and provided a promising target to rationally design TAG molecular with the desired characteristics.

An emerging simple and effective approach to increase the productivity of thraustochytrids microbial lipids by regulating glycolysis process and triacylglycerols’ decomposition

Background The oleaginous microorganism Schizochytrium sp. is widely used in scientific research and commercial lipid production processes. However, low glucose-to-lipid conversion rate (GLCR) and low lipid productivity of Schizochytrium sp. restrict the feasibility of its use. Results Orlistat is a lipase inhibitor, which avoids triacylglycerols (TAGs) from hydrolysis by lipase. TAGs are the main storage forms of fatty acids in Schizochytrium sp. In this study, the usage of orlistat increased the GLCR by 21.88% in the middle stage of fermentation. Whereas the productivity of lipid increased 1.34 times reaching 0.73 g/L/h, the saturated fatty acid and polyunsaturated fatty acid yield increased from 21.2 and 39.1 to 34.9 and 48.5 g/L, respectively, indicating the advantages of using a lipase inhibitor in microbial lipids fermentation. Similarly, the system was also successful in Thraustochytrid Aurantiochytrium. The metabolic regulatory mechanisms stimulated by orlistat in Schizochytrium sp. were further investigated using transcriptomics and metabolomics. The results showed that orlistat redistributed carbon allocation and enhanced the energy supply when inhibiting the TAGs’ degradation pathway. Therefore, lipase in Schizochytrium sp. prefers to hydrolyze saturated fatty acid TAGs into the β-oxidation pathway. Conclusions This study provides a simple and effective approach to improve lipid production, and makes us understand the mechanism of lipid accumulation and decomposition in Schizochytrium sp., offering new guidance for the exploitation of oleaginous microorganisms.

Simultaneous production of DHA and squalene from Aurantiochytrium sp. grown on forest biomass hydrolysates

Background Recent evidence points to the nutritional importance of docosahexaenoic acid (DHA) in the human diet. Thraustochytrids are heterotrophic marine oleaginous microorganisms capable of synthesizing high amounts of DHA, as well as other nutraceutical compounds such as squalene, in their cellular compartment. Squalene is a natural triterpene and an important biosynthetic precursor to all human steroids. It has a wide range of applications in the cosmetic and pharmaceutical industries, with benefits that include boosting immunity and antioxidant activity. Apart from its nutritional quality, it can also be utilized for high-grade bio-jet fuel by catalytic conversion. Results In the present study, the potential of thraustochytrid strain Aurantiochytrium sp. T66 to produce DHA and squalene was evaluated. When the strain was cultivated on organosolv-pretreated birch hydrolysate (30 g/L glucose) in flask, it resulted in 10.39 g/L of cell dry weight and 4.98 g/L of total lipids, of which 25.98% was DHA. In contrast, when the strain was grown in a bioreactor, cell dry weight, total lipid, and DHA increased to 11.24 g/L, 5.90 g/L, and 35.76%, respectively. The maximum squalene yield was 69.31 mg/gCDW (0.72 g/L) when the strain was cultivated in flask, but it increased to 88.47 mg/gCDW (1.0 g/L), when cultivation shifted to a bioreactor. Conclusions This is the first report demonstrating the utilization of low cost non-edible lignocellulosic feedstock to cultivate the marine oleaginous microorganism Aurantiochytrium sp. for the production of nutraceutical vital compounds. Owing to the simultaneous generation of DHA and squalene, the strain is suitable for industrial-scale production of nutraceuticals.

Use of a novel oleaginous microorganism as a potential source of lipids for weanling pigs1,2

Weanling pigs are at risk of succumbing to illness due to an immature immune system and insufficient supply of available energy at the time of weaning. This study was aimed at determining whether oleaginous bacteria could serve as a source of lipids to weanling pigs. Weanling pigs were provided a daily dose of 1×109 colony fomring unit (CFU) = kg−1 of the novel oleaginous Enterobacter cloacae strain JD6301 or JD8715 (which is a variant form of JD6301 capable of producing extracellular triglycerides) via oral gavage for 5 d. Serum was collected every 6 h and intestinal samples were collected at 6 d. Providing pigs with JD6301 or JD8715 significantly increased serum concentrations of triglycerides and non-esterified fatty acids (NEFA) within 72 h. Additionally, the JD6301 and JD8715 strains were able to survive within the gastrointestinal tract throughout the duration of the study. These results suggest that providing Enterobacter cloacae can increase the serum lipids in the pigs, thus potentially providing an additional source of energy to animals during times of stress. This could potentially help improve the metabolic response of animals during times of stress.

PRODUCTION OF DOCOSAHEXAENOIC ACID (DHA) FROM Thraustochytrium sp. ATCC 26185 USING DIFFERENTS NITROGEN CONCENTRATIONS

Polyunsaturated fatty acids (PUFAs) of the type ω3 and ω6 play important physiological functions in human organism, since they are components of cell membranes and brain cells; they decrease the levels of triglycerides and can prevent the incidence of coronary heart disease. Various parameters, including concentration of the nitrogen source in the cultivation of oleaginous microorganisms have been reported to be essential in the biosynthesis and accumulation of PUFAs. The objective of this work is to study the effect of different concentrations of total nitrogen (TN) in the production of PUFAs, especially DHA, from Thraustochytrium sp. ATCC 26185. The concentrations of TN evaluated were 2.4 and 0.8 g/L (batch) and 0.009 g/L (hourly) under fed-batch process. The content of cell biomass, glucose consumption, TN and production of PUFAs was determined. The major composition of the PUFAs in Thraustochytrium sp. ATCC 26185 cell biomass were DPA ω6 (21-25%) and DHA (69-73%), regardless of the type and time of culture. The maximum cell concentration obtained (30.2 g/L) was using 2.4 g/L TN in 168 h of culture. With this same concentration of TN it was possible to produce the highest concentration of DHA (1.16 g/L) in 120 h of culture, demonstrating that the growth of Thraustochytrium sp. ATCC 26185 and yield of PUFAs are dependent on the concentration TN source available for consumption of this oleaginous microorganism, as well as culture time.

PRODUCTION OF DOCOSAHEXAENOIC ACID (DHA) FROM Thraustochytrium sp. ATCC 26185 USING DIFFERENTS NITROGEN CONCENTRATIONS

In this work it was studied the polyunsaturated fatty acids (PUFAs) production, especially DHA, from Thraustochytrium sp. ATCC 26185, under different total nitrogen (TN) availability. Three different TN conditions were evaluated: two with initial concentrations of 2.4 g/L and 0.8 g/L, and the third in a fed-batch process with a rate of 0.009 g/L.h. For each experiment the biomass, glucose, TN and PUFAs were determined. The major composition of the PUFAs in Thraustochytrium sp. ATCC 26185 cell biomass were DPA ω6 (21-25 %) and DHA (69-73 %), regardless of the type and time of culture. The maximum cell concentration (30.2 g/L) was obtained using 2.4 g/L TN in 168 h of culture. With this same concentration of TN it was possible to produce the highest concentration of DHA (1.16 g/L) in 120 h of culture, demonstrating that the growth of Thraustochytrium sp. ATCC 26185 and yield of PUFAs are dependent on the source concentration of TN available for consumption of this oleaginous microorganism, as well as culture time.