Aurantiochytrium Limacinum Research Articles

Transcriptional, post-transcriptional, and post-translational regulation of polyunsaturated fatty acid synthase genes in Aurantiochytrium limacinum strain BL10: Responses to nitrogen starvation

Under nitrogen deficient conditions, the Aurantiochytrium limacinum strain BL10 greatly increases the production of docosahexaenoic acid (DHA) and n-6 docosapentaenoic acid. Researchers have yet to elucidate the mechanism by which BL10 promotes the activity of polyunsaturated fatty acid synthase (Pfa), which plays a key role in the synthesis of polyunsaturated fatty acid (PUFA). Analysis in the current study revealed that in nitrogen-depleted environments, BL10 boosts the transcription and synthesis of proteins by facilitating the expression of pfa genes via transcriptional regulation. It was also determined that BL10 adjusts the lengths of the 5′- and 3′-untranslated regions (suggesting post-transcriptional regulation) and modifies the ratio of two Pfa1 isoforms to favor PUFA production via post-translational regulation (ubiquitination). These findings clarify the exceptional DHA production of BL10 and provide additional insights into the regulatory mechanisms of PUFA biosynthesis in Aurantiochytrium.

Biochemical characterization of lipid metabolic genes of Aurantiochytrium limacinum

Docosahexaenoic acid (DHA) is known to have numerous health benefits and immense dietary value. There is a pressing need to have a deeper understanding of DHA metabolism. Acyl CoA: Diacylglycerol Acyltransferase (DGAT) is an important enzyme of lipid anabolism and an essential piece of the puzzle. Aurantiochytrium limacinum, a primary producer of DHA, is a good model for studying DHA metabolism. Thus, we aimed to investigate important lipid metabolic genes from A. limacinum. We cloned four putative DGATs (DGAT2a, DGAT2b, DGAT2c, and DGAT2d) from A. limacinum and performed detailed in vivo and in vitro characterization. Functional characterization showed that not all the studied genes exhibited DGAT activity. DGAT2a and DGAT2d conferred DGAT activity whereas DGAT2b showed wax synthase (WS) activity and DGAT2c showed dual function of both WS and DGAT. Based on their identified function, DGAT2b and DGAT2c were renamed as AlWS and AlWS/DGAT respectively. DGAT2a was found to exhibit a preference for DHA as a substrate. DGAT2d was found to have robust activity and emerged as a promising candidate for genetic engineering aimed at increasing oil yield. The study enriches our knowledge of lipid biosynthetic enzymes in A. limacinum, which can be utilized to design suitable application strategies.

The protist Aurantiochytrium has universal subtelomeric rDNAs and is a host for mirusviruses

Viruses are the most abundant biological entities in the world's oceans, where they play important ecological and biogeochemical roles. Metagenomics is revealing new groups of eukaryotic viruses, although disconnected from known hosts. Among these are the recently described mirusviruses, which share some similarities with herpesviruses.1 50 years ago, "herpes-type" viral particles2 were found in a thraustochytrid member of the labyrinthulomycetes, a diverse group of abundant and ecologically important marine eukaryotes,3,4 but could not be further characterized by methods then available. Long-read sequencing has allowed us to connect the biology of mirusviruses and thraustochytrids. We sequenced the genome of the genetically tractable model thraustochytrid Aurantiochytrium limacinum ATCC MYA-1381 and found that its 26 linear chromosomes have an extraordinary configuration. Subtelomeric ribosomal DNAs (rDNAs) found at all chromosome ends are interspersed with long repeated sequence elements denoted as long repeated-telomere and rDNA spacers (LORE-TEARS). We identified two genomic elements that are related to mirusvirus genomes. The first is a ∼300-kbp episome (circular element 1 [CE1]) present at a high copy number. Strikingly, the second, distinct, mirusvirus-like element is integrated between two sets of rDNAs and LORE-TEARS at the left end of chromosome 15 (LE-Chr15). Similar to metagenomically derived mirusviruses, these putative A.limacinum mirusviruses have a virion module related to that of herpesviruses along with an informational module related to nucleocytoplasmic large DNA viruses (NCLDVs). CE1 and LE-Chr15 bear striking similarities to episomal and endogenous latent forms of herpesviruses, respectively, and open new avenues of research into marine virus-host interactions.

Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186.

Since the global market for sterols and vitamin D are grown with a high compound annual growth rate, a sustainable source of these compounds is required to keep up with the increasing demand. Thraustochytrid is a marine oleaginous microorganism that can synthesize several sterols, which are stored as SE in lipid droplets. DGAT2C is an unconventional SE synthase specific to thraustochytrids. Although the primary structure of DGAT2C shows high similarities with that of DGAT, DGAT2C utilizes sterol as an acceptor substrate instead of diacylglycerol. In this study, we examined more detailed enzymatic properties, intracellular localization, and structure-activity relationship of DGAT2C. Furthermore, we successfully developed a method to increase sterol and provitamin D3 productivity of thraustochytrid by more than threefold in the process of elucidating the function of the DGAT2C-specific N-terminal region. Our findings could lead to sustainable sterol and vitamin D production using thraustochytrid.

Identification and Functional Analysis of Two Novel Genes-Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)-from Aurantiochytrium limacinum Significantly Enhance De Novo β-Carotene Biosynthesis in Escherichia coli.

Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo β-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of β-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in β-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, β-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.

Utilizing lipidomics and fatty acid synthase inhibitors to explore lipid accumulation in two thraustochytrid species

Thraustochytrids are marine protists with an excellent ability to produce health-beneficial docosahexaenoic acid (DHA)-rich lipids. Since the demand for DHA sources for food and feed is increasing, a deeper understanding of the potential to enhance the DHA-production in thraustochytrids is important for a sustainable DHA supply. In most thraustochytrids, including the strains used in this work, DHA is synthesized by a polyketide synthase-like pathway (PKS), and not through the common fatty acid synthetase (FAS) pathway. Both pathways use the same precursors. The aim of this study was to determine if inhibiting the production of fatty acids (FA) through the FAS pathway under nitrogen starvation would result in higher net DHA production rates measured as DHA content, and if the channeling of the synthesized fatty acids to different lipids species is similar in two different thraustochytrid strains. We found that the DHA production rates in Aurantiochytrium limacinum SR21 and Aurantiochytrium sp. T66 were not affected when treated with the FAS-inhibitor cerulenin, despite a reduction of FAS-derived FAs. This indicates that precursor availability is less likely to be the limiting factor for DHA synthesis. Still, cerulenin-treatment in the lipid accumulation phase resulted in higher concentrations of the triacylglycerol (TAG) TG(22:6/22:6/22:6) than in the untreated cells after three hours, demonstrating that FAS inhibition is a possible strategy to enhance the content of DHA-rich lipids. Moreover, the relatively high abundance of the diacylglycerol DG(22:6/22:6) suggests the presence of an obstacle for using DG(22:6/22:6) as substrate to synthesize DHA-rich TAGs, a step that potentially can be improved by metabolic engineering.

Saturated and Polyunsaturated Fatty Acids Production by Aurantiochytrium limacinum PKU#Mn4 on Enteromorpha Hydrolysate.

Thraustochytrids are unicellular marine heterotrophic protists, which have recently shown a promising ability to produce omega-3 fatty acids from lignocellulosic hydrolysates and wastewaters. Here we studied the biorefinery potential of the dilute acid-pretreated marine macroalgae (Enteromorpha) in comparison with glucose via fermentation using a previously isolated thraustochytrid strain (Aurantiochytrium limacinum PKU#Mn4). The total reducing sugars in the Enteromorpha hydrolysate accounted for 43.93% of the dry cell weight (DCW). The strain was capable of producing the highest DCW (4.32 ± 0.09 g/L) and total fatty acids (TFA) content (0.65 ± 0.03 g/L) in the medium containing 100 g/L of hydrolysate. The maximum TFA yields of 0.164 ± 0.160 g/g DCW and 0.196 ± 0.010 g/g DCW were achieved at 80 g/L of hydrolysate and 40 g/L of glucose in the fermentation medium, respectively. Compositional analysis of TFA revealed the production of equivalent fractions (% TFA) of saturated and polyunsaturated fatty acids in hydrolysate or glucose medium. Furthermore, the strain yielded a much higher fraction (2.61-3.22%) of eicosapentaenoic acid (C20:5n-3) in the hydrolysate medium than that (0.25-0.49%) in the glucose medium. Overall, our findings suggest that Enteromorpha hydrolysate can be a potential natural substrate in the fermentative production of high-value fatty acids by thraustochytrids.

Simultaneous Imaging and Characterization of Polyunsaturated Fatty Acids, Carotenoids, and Microcrystalline Guanine in Single Aurantiochytrium limacinum Cells with Linear and Nonlinear Raman Microspectroscopy.

Thraustochytrids are heterotrophic marine protists known for their high production capacity of various compounds with health benefits, such as polyunsaturated fatty acids and carotenoids. Although much effort has been focused on developing optimal cultivation methods for efficient microbial production, these high-value compounds and their interrelationships are not well understood at the single-cell level. Here we used spontaneous (linear) Raman and multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy to visualize and characterize lipids (e.g., docosahexaenoic acid) and carotenoids (e.g., astaxanthin) accumulated in single living Aurantiochytrium limacinum cells. Spontaneous Raman imaging with the help of multivariate curve resolution-alternating least-squares enabled us to make unambiguous assignments of the molecular components we detected and derive their intracellular distributions separately. Near-IR excited CARS imaging yielded the Raman images at least an order of magnitude faster than spontaneous Raman imaging, with suppressed contributions of carotenoids. As the culture time increased from 2 to 5 days, the lipid amount increased by a factor of ∼7, whereas the carotenoid amount did not change significantly. Furthermore, we observed a highly localized component in A. limacinum cells. This component was found to be mixed crystals of guanine and other purine derivatives. The present study demonstrates the potential of the linear-nonlinear Raman hybrid approach that allows for accurate molecular identification and fast imaging in a label-free manner to link information derived from single cells with strategies for mass culture of useful thraustochytrids.

Production of docosahexaenoic acid from spruce sugars using Aurantiochytrium limacinum

In this study lignocellulosic sugars from Norway spruce were used for production of docosahexaenoic acid (DHA) by the marine thraustochytrid Aurantiochytrium limacinum SR21. Enzymatically prepared spruce hydrolysate was combined with a complex nitrogen source and different amounts of salts. Shake flask batch cultivations revealed that addition of extra salts was not needed for optimal growth. Upscaling to fed-batch bioreactors yielded up to 55 g/L cell dry mass and a total fatty acid content of 44% (w/w) out of which 1/3 was DHA. Fourier transform infrared spectroscopy was successfully applied as a rapid method for monitoring lipid accumulation in A. limacinum SR21. Thus, this proof-of-principle study clearly demonstrates that crude spruce hydrolysates can be directly used as a novel and sustainable resource for production of DHA.

Horizontal Gene Transfer and Fusion Spread Carotenogenesis Among Diverse Heterotrophic Protists.

Thraustochytrids (phylum: Labyrinthulomycota) are nonphotosynthetic marine protists. Some thraustochytrids have crtIBY, a trifunctional fusion gene encoding a protein capable of β-carotene biosynthesis from geranylgeranyl pyrophosphate. Here we show that crtIBY is essential in, and encodes the sole pathway for, carotenoid biosynthesis in the thraustochytrid Aurantiochytrium limacinum ATCC MYA-1381. We explore the evolutionary origins of CrtIBY and discover that the closest related protein domains are present in a small but diverse group of other heterotrophic protists, including the apusomonad Thecamonas trahens and the dinoflagellates Oxyrrhis marina and Noctiluca scintillans. Each organism within this cluster also contains one or more β-carotene 15-15' oxygenase genes (blh and rpe65), suggesting that the acquisition of β-carotene biosynthesis genes may have been related to the production of retinal. Our findings support a novel origin of eukaryotic (apo)carotenoid biosynthesis by horizontal gene transfer from Actinobacteria, Bacteroidetes, and/or Archaea. This reveals a remarkable case of parallel evolution of eukaryotic (apo)carotenogenesis in divergent protistan lineages by repeated gene transfers.

Improvement of Glucose Intolerance in Asian-Type Diabetes Model Mice Using Pentadecyl, Pentadecanoic Acid-Based Triglyceride

Even-chain saturated fatty acid concentrations are associated with an increased risk of coronary heart disease, whereas oddchain saturated fatty acid concentrations, such as pentadecanoic acid (15:0) and heptadecanoic acid (17:0), are associated with a decreased risk. Similarly, even-chain saturated fatty acids are positively associated with type 2 diabetes (T2D), whereas odd-chain saturated fatty acids are thought to be inversely associated with T2D. We purified pentadecyl, pentadecanoic acidbased triglycerides from Aurantiochytrium limacinum, a type of marine microalgae, and demonstrated the effect of pentadecyl on insulin secretion both in vitro and in vivo. Pentadecyl increased the cell viability of immortalized mouse pancreatic beta cells in a dose-dependent manner and induced high glucose-stimulated insulin secretion in the cells. Pancreatic beta cellspecific CDKAL1-deficient mice, a model of Asian-type T2D, were given pentadecyl with a low- or high-fat diet for 9 weeks, and glucose tolerance tests were performed. Pentadecyl improved the glucose intolerance of the mice and decreased the expressions of ER stress-related genes in the islets of the mice. These results suggest that pentadecyl can improve insulin secretion through the reduction of ER stress in patients with Asian-type T2D.

Transcriptome analysis of Aurantiochytrium limacinum under low salt conditions.

Aurantiochytrium limacinum can accumulate high amounts of omega-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). Although salinity affects the DHA content, its impact on the metabolic pathway responsible for DHA production in A. limacinum is not completely understood. To address this issue, we investigated the transcriptional profile of A. limacinum under hypoosmotic stress. We first cultured A. limacinum under typical and low salinity for RNA sequencing, respectively. Transcriptome analyses revealed that 933 genes exhibited significant changes in expression under hypoosmotic conditions, of which 81.4% were downregulated. Strikingly, A. limacinum downregulated genes related to polyketide synthesis and fatty acid synthase pathways, while upregulating β-oxidation-related genes. In accordance with this, DHA production significantly decreased under hypoosmotic conditions, while antioxidant-related genes were significantly upregulated. Considering that β-oxidation of fatty acids generates energy and reactive oxygen species (ROS), our results suggest that A. limacinum utilizes fatty acids for energy to survive under hypoosmotic conditions and detoxifies ROS using antioxidant systems.

Improvement of Astaxanthin Production in Aurantiochytrium limacinum by Overexpression of the Beta-Carotene Hydroxylase Gene.

Aurantiochytrium limacinum is a heterotrophic eukaryotic microorganism that can accumulate high levels of commercial products such as astaxanthin and docosahexaenoic acid. Due to its rapid growth and relatively simple extraction method, A. limacinum is considered a promising astaxanthin resource to replace the conventional microalgal production. However, the astaxanthin biosynthetic process in A. limacinum remains incompletely understood, especially in those catalysed by β-carotene hydroxylase (CrtZ) and ketolase. In this study, we overexpressed a crtZ candidate gene to increase astaxanthin production and expand our understanding of the conversion from beta-carotene to astaxanthin. The resultant transformant AlcrtZ#10 cultivated for 5days showed a significant increase in astaxanthin production per culture (2.8-fold) and per cell (4.5-fold) compared with that of the wild-type strain. Strikingly, longer light exposure increased astaxanthin production and decreased the beta-carotene content in the wild-type strain, suggesting that light exposure duration is important for astaxanthin production in A. limacinum. Among several predicted intermediates, furthermore, the cantaxanthin produced from β-carotene by ketolase activity were enhanced in the transformant AlcrtZ#10. Although the further investigation is needed, this result suggested that the main route of astaxanthin was via cantaxanthin. Thus, our findings will be valuable not only for its application, but also for understanding the astaxanthin biosynthetic process in A. limacinum.

Proteomic and lipidomic analyses of lipid droplets in Aurantiochytrium limacinum ATCC MYA-1381

Aurantiochytrium limacinum is a heterotrophic unicellular organism that develops cytosolic lipid droplets that store triacylglycerols (TGs) with a high docosahexaenoic acid (C22:6) content. Although the information on protein and lipid compositions in lipid droplets is useful for the understanding of storage lipid metabolism, it is not well known in this organism. In the present study, we purified lipid droplets by mild disruption of the cells using a hypo-osmotic buffer containing 0.05 % or 0.2 % NP-40 and analyzed the proteomics and lipidomics. In total, 29 putative lipid droplet-localizing proteins were identified. Approximately half of the lipid droplet protein content [54.0 % (w/w)] was accounted for by thraustochytrid-specific lipid droplet protein 1 (TLDP1), which might be a structural protein in the lipid droplet. The proteins included acyl-CoA synthetase (ACS), sterol 24-C methyltransferase (SMT1), diacylglycerol acyltransferase 2 (DGAT2), and heat-shock protein 70 kDa (HSP70), which are often found in lipid droplets isolated from other organisms. We also found putative orthologs of a lipid droplet-associated hydrolase which catalyzes the hydrolysis of cholesterol esters in macrophages and a monoglyceride lipase that has been identified in mammalian lipid droplets. The lipidomics showed that the major unsaturated TGs were TG60:12 [palmitic acid (C16:0)/C22:6/C22:6] and TG66:18 (C22:6/C22:6/C22:6). Phytosterol ester was more abundant in the lipid droplet fraction than the nonlipid droplet fraction. The accumulation of phytosterol ester in the lipid droplet might have a causal relationship to the localization of SMT1, which is considered to act in the phytosterol biosynthesis pathway. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were present, but the lipid droplet fraction was deficient in lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Through the proteome and lipidome analyses, the characteristics of proteins and lipids in A. limacinum lipid droplets were shown.

Significance of mitochondrial fatty acid β-oxidation for the survivability of Aurantiochytrium limacinum ATCC MYA-1381 during sugar starvation.

Thraustochytrids are marine protists that accumulate large amounts of palmitic acid and docosahexaenoic acid in lipid droplets. Random insertional mutagenesis was adopted for Aurantiochytrium limacinum ATCC MYA-1381 to search for genes that regulate lipid metabolism in thraustochytrids. A mutant strain, M17, was selected because of its significant decrease in myristic acid, palmitic acid, and triacylglycerol contents and cell growth defect. Genome analysis revealed that the gene encoding for mitochondrial electron-transfer flavoprotein ubiquinone oxidoreductase (ETFQO) was lacking in the M17 strain. This mutant strain exhibited a growth defect at the stationary phase, possibly due to stagnation of mitochondrial fatty acid β-oxidation and branched-chain amino acid degradation, both of which were caused by lack of ETFQO. This study shows the usability of random insertional mutagenesis to obtain mutants of lipid metabolism in A. limacinum and clarifies that ETFQO is integral for survival under sugar starvation in A. limacinum.

Docosahexaenoic acid (DHA) production by Aurantiochytrium limacinum using cassava pulp hydrolysate as an alternative low-cost carbon source

Cost of nutrients is one of the major contributors to the production cost of docosahexaenoic acid (DHA) by thraustochytrids, and this remains the main challenge for economical and sustainable production of DHA. In the present study, cassava pulp (CP) was investigated as an alternative low-cost carbon source for DHA production by Aurantiochytrium limacinum SR21. Cultivation conditions, i.e., salinity level, type of nitrogen source, and concentrations of glucose and nitrogen sources, as well as pH, were optimized for cell growth. CP was enzymatically hydrolyzed, and used as the base medium, supplemented with artificial seawater salts, for DHA production under fed-batch cultivation. A. limacinum grew well at 18 g/L of NaCl (equivalent to 50% salinity level), and initial glucose concentration of 64 g/L, initial yeast extract concentration of 5 g/L, and pH 7.5 were optimum for cell growth. Fed-batch cultivation, with dissolved oxygen (DO) controlled at 10%, using CP-based medium as the carbon source gave a final cell dry mass of 26.3 ± 2.0 g/L, with 15.5 ± 0.1 g/L of lipid. DHA content in the lipid was 37.4 ± 1.5%, equivalent to 5.8 ± 0.3 g/L, and DHA productivity was 960 mg/(L·d). Medium-based economic analysis showed that an economic yield (EY) of this process was 0.86, which was higher than the 0.81 obtained using glucose as the substrate, assuming the same process and conditions. Overall, the results reveal that CP is a highly feasible feedstock for DHA production by A. limacinum SR21.

Influence of Dietary Algae Meal on Lipid Oxidation and Volatile Profile of Meat from Lambs with Competent Reticular Groove Reflex.

Dietary lipid sources influence intramuscular fatty acid composition, which in turn may affect the volatile profile of meat. The aim of this work was to investigate the effects of marine algae supplementation (Aurantiochytrium limacinum) on volatile compounds of cooked lamb meat. Forty-eight lambs with 42 days of age were divided into three groups: lambs fed a conventional diet without algae meal supplementation (NOALG), lambs with competent reticular groove reflex (RGR) fed the same diet supplemented with 2.5% marine algae meal mixed in the concentrate (ALGCON), and lambs with competent RGR, receiving the same diet and fed with 2.5% marine algae meal in a milk replacer to bypass the rumen (ALGMILK). Lipid and protein oxidation in raw meat was assessed and volatile compounds in grilled meat were determined. The highest and lowest lipid oxidations were observed in the ALGMILK and NOALG groups, respectively. Protein oxidation was unaffected. Out of 56 identified compounds, 12 volatiles significantly increased in both algae groups and 6 of them exclusively in the ALGCON treatment. Algae meal supplementation and its form of administration, either protected or not from rumen degradation, are important factors to consider in lipid oxidation and the aromatic profile of lamb meat.

Transcriptional responses of Aurantiochytrium limacinum under light conditions.

Astaxanthin-producing protist Aurantiochytrium limacinum can accumulate higher amounts of astaxanthin under light conditions; however, little is known about the impact of light exposure on its metabolism. Here, we investigated the transcriptional profile of A. limacinum under light conditions. Transcriptomic analyses revealed that 962 genes of A. limacinum showed a significant change in expression under light conditions, most of which (94.5%) were downregulated. Furthermore, gene ontology enrichment analysis indicated that A. limacinum mainly downregulated genes associated with cell motility, proliferation and gene expression processes, whose activities depend on ATP as an energy source. Additionally, the quantification of carotenoid and its transcripts suggested that β-carotene and astaxanthin biosynthesis pathways were rate-limiting and tightly regulated steps, respectively. In comparison, these processes were enhanced under light conditions. Considering that astaxanthin accumulation was highly correlated with reactive oxygen species (ROS) levels in microalgae, our results suggest that A. limacinum reduces ATP consumption to decrease the occurrence of ROS in mitochondria while accumulating astaxanthin to prevent ROS damage. This study provides novel insights into the impact of light exposure on A. limacinum metabolism, thereby facilitating a complete understanding of this protist for efficient astaxanthin production.

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach.

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.

Overexpression of Two New Acyl-CoA:Diacylglycerol Acyltransferase 2-Like Acyl-CoA:Sterol Acyltransferases Enhanced Squalene Accumulation in Aurantiochytrium limacinum.

Thraustochytrids are heterotrophic marine eukaryotes known to accumulate large amounts of triacylglycerols, and they also synthesize terpenoids like carotenoids and squalene, which all have an increasing market demand. However, a more extensive knowledge of the lipid metabolism is needed to develop thraustochytrids for profitable biomanufacturing. In this study, two putative type-2 Acyl-CoA:diacylglycerol acyltransferases (DGAT2) genes of Aurantiochytrium sp. T66, T66ASATa, and T66ASATb, and their homologs in Aurantiochytrium limacinum SR21, AlASATa and AlASATb, were characterized. In A. limacinum SR21, genomic knockout of AlASATb reduced the amount of the steryl esters of palmitic acid, SE (16:0), and docosahexaenoic acid, SE (22:6). The double mutant of AlASATa and AlASATb produced even less of these steryl esters. The expression and overexpression of T66ASATb and AlASATb, respectively, enhanced SE (16:0) and SE (22:6) production more significantly than those of T66ASATa and AlASATa. In contrast, these mutations did not significantly change the level of triacylglycerols or other lipid classes. The results suggest that the four genes encoded proteins possessing acyl-CoA:sterol acyltransferase (ASAT) activity synthesizing both SE (16:0) and SE (22:6), but with the contribution from AlASATb and T66ASATb being more important than that of AlASATa and T66ASATa. Furthermore, the expression and overexpression of T66ASATb and AlASATb enhanced squalene accumulation in SR21 by up to 88%. The discovery highlights the functional diversity of DGAT2-like proteins and provides valuable information on steryl ester and squalene synthesis in thraustochytrids, paving the way to enhance squalene production through metabolic engineering.