Aurantiochytrium Sp Research Articles

Utilization of low-cost substrates for the production of high biomass, lipid and docosahexaenoic acid (DHA) using local native strain Aurantiochytrium sp. YB-05

This study explores low-cost substrates for the production of high biomass, lipid and DHA by local native strain of thraustochytrid that was isolated from decaying leaves of Avicennia marina collected from Yanbu mangroves along the Red Sea coast of Saudi Arabia. Molecular phylogenetic analyses based on 18S rDNA sequences placed the thraustochytrid strain (YB-05) in the Aurantiochytrium. The studied strain was grown on three different types of raw material available in the local market and compared to pure glucose for high biomass, lipid and DHA production. Aurantiochytrium sp. YB-05 was grown on: date fruit, glycerol, cane molasses and pure glucose at four concentrations: 10, 20, 40 and 80 g/L. Produced dry cell weight increased as the carbon sources: date fruit, molasses and glucose increased from 10 to 80 g/L. Both date fruit and molasses gave a higher biomass than pure glucose and glycerol at 40 and 80 g/L concentration, while glucose gave higher biomass at 10 and 20 g/L than the other three carbon sources. Date fruit gave the highest biomass (42.6 g/L) at the 80 g/L concentration and also increased the level of DPA (decosapentaneoic acid) and DHA from 0.64 and 4.89% TFA (total fatty acids) with glucose to 7.13 and 24.85% with date fruit respectively. Ten g/L glycerol gave the highest percentage of lipids (85.6% of the biomass), followed by 40 g/L glucose (80.1%), 20 g/L glycerol (70%), 80 g/L glucose (59.3%) and 80 g/L date fruit (56.4%). The percentage of lipid in the biomass increased with increasing the weight of date fruit and molasses from 10 g/L (24.7% and 15.6% respectively) to 80 g/L (56.4% and 45.7% respectively). This study shed a light on the potential use of date fruits for the production of value-added products using microbes. Saudi Arabia is the second largest date producer in the world after Egypt. The present research also studied the effect of four types of oily seeds in a combination with date fruit on the production oil, DHA and the major fatty acids produced by Aurantiochytrium sp. YB-05. The fatty acid profile of Aurantiochytrium sp. YB-05 grown on date palm, pure glucose and seeds were determined.

Strategic Development of Aurantiochytrium sp. Mutants With Superior Oxidative Stress Tolerance and Glucose-6-Phosphate Dehydrogenase Activity for Enhanced DHA Production Through Plasma Mutagenesis Coupled With Chemical Screening.

Thraustochytrids, such as Aurantiochytrium and Schizochytrium, have been shown as a promising sustainable alternative to fish oil due to its ability to accumulate a high level of docosahexaenoic acid (DHA) from its total fatty acids. However, the low DHA volumetric yield by most of the wild type (WT) strain of thraustochytrids which probably be caused by the low oxidative stress tolerance as well as a limited supply of key precursors for DHA biosynthesis has restricted its application for industrial application. Thus, to enhance the DHA production, we aimed to generate Aurantiochytrium SW1 mutant with high tolerance toward oxidative stress and high glucose-6 phosphate dehydrogenase (G6PDH) activities through strategic plasma mutagenesis coupled with chemical screening. The WT strain (Aurantiochytrium sp. SW1) was initially exposed to plasma radiation and was further challenged with zeocin and polydatin, generating a mutant (YHPM1) with a 30, 65, and 80% higher overall biomass, lipid, and DHA production in comparison with the parental strains, respectively. Further analysis showed that the superior growth, lipid, and DHA biosynthesis of the YHMP1 were attributed not only to the higher G6PDH and enzymes involved in the oxidative defense such as superoxide dismutase (SOD) and catalase (CAT) but also to other key metabolic enzymes involved in lipid biosynthesis. This study provides an effective approach in developing the Aurantiochytrium sp. mutant with superior DHA production capacity that has the potential for industrial applications.

Development of a fed-batch fermentation with stepwise aeration to enhance docosahexaenoic acid and carotenoid content in Aurantiochytrium sp. FIKU018

Development of a fed-batch fermentation with stepwise aeration to enhance docosahexaenoic acid and carotenoid content in Aurantiochytrium sp. FIKU018

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.

Utilization of Sugarcane Bagasse as a Substrate for Lipid Production by Aurantiochytrium sp.

Thraustochytrid, Aurantiochytrium sp., produces various lipids such as polyunsaturated and saturated fatty acids, carotenoids, and other hydrocarbons, which are useful in the fields of health foods, cosmetics, fine chemicals, and biofuels. Lignocellulosic biomass, which is abundant and cheap, is a promising feedstock for producing cheaper bulk and high-value-added products using Aurantiochytrium sp. However, the steam explosion of lignocellulosic biomass for efficient enzymatic saccharification generates substances that inhibit the growth of microorganisms. In this study, the inhibitory activities of these by-products on the growth and lipid production of Aurantiochytrium sp. were investigated. Aurantiochytrium sp. was found to be highly sensitive to furfural and vanillin and moderately sensitive to 5-hydroxymethylfurfural and syringaldehyde. Washing steam-exploded bagasse with water, followed by activated charcoal treatment, significantly reduced furfural, which was a major inhibitory component in the saccharified solution.

A Two-Stage Adaptive Laboratory Evolution Strategy to Enhance Docosahexaenoic Acid Synthesis in Oleaginous Thraustochytrid.

Thraustochytrid is a promising algal oil resource with the potential to meet the demand for docosahexaenoic acid (DHA). However, oils with high DHA content produced by genetic modified thraustochytrids are not accepted by the food and pharmaceutical industries in many countries. Therefore, in order to obtain non-transgenic strains with high DHA content, a two-stage adaptive laboratory evolution (ALE) strategy was applied to the thraustochytrid Aurantiochytrium sp. Heavy-ion irradiation technique was first used before the ALE to increase the genetic diversity of strains, and then two-step ALE: low temperature based ALE and ACCase inhibitor quizalofop-p-ethyl based ALE were employed in enhancing the DHA production. Using this strategy, the end-point strain E-81 with a DHA content 51% higher than that of the parental strain was obtained. The performance of E-81 strain was further analyzed by component analysis and quantitative real-time PCR. The results showed that the enhanced in lipid content was due to the up-regulated expression of key enzymes in lipid accumulation, while the increase in DHA content was due to the increased transcriptional levels of polyunsaturated fatty acid synthase. This study demonstrated a non-genetic approach to enhance lipid and DHA content in non-model industrial oleaginous strains.

Evaluating the Potential of the Defatted By-Product of Aurantiochytrium sp. Industrial Cultivation as a Functional Food.

While Aurantiochytrium sp. is an increasingly popular source of polyunsaturated fatty acids (PUFAs), its extraction generates high amounts of waste, including the spent, defatted residue. The composition and bioactivities of this by-product could prove to be a major part of the sustainable valorisation of this organism within the framework of a circular economy. In this study, the defatted biomass of commercial Aurantiochytrium sp. was nutritionally characterised, and its amino acid profile was detailed. Additionally, the antioxidant and prebiotic potentials of an enzymatically digested sample of defatted Aurantiochytrium sp. were evaluated under a set of miniaturised in vitro assays. The nutritional profile of the spent Aurantiochytrium biomass revealed a protein and dietary-fibre rich product, with values reaching 26.7% and 31.0% for each, respectively. It also held high concentrations of glutamic and aspartic acid, as well as a favourable lysine/arginine ratio of 3.73. The digested samples demonstrated significant Weissela cibaria and Bifidobacterium bifidum growth-enhancing potential. Residual ferric reducing antioxidant power (FRAP) activity was likely attributed to antioxidant amino acids or peptides. The study demonstrated that some of the nutritional and functional potential that reside in the defatted Aurantiochytrium sp. waste encourages additional studies and the development of food supplements employing this resource’s by-products under a biorefinery framework.

Isolation and characterization of the ω3-docosapentaenoic acid-producing microorganism Aurantiochytrium sp. T7

ω3-Docosapentaenoic acid (ω3-DPA), an ω3-polyunsaturated fatty acid (ω3-PUFA), is expected to have beneficial physiological functions to humans; however, because of its rarity in nature, it has not been fully analyzed. We isolated an ω3-DPA producing microorganism strain T7 from brackish areas in Japan. Although most oleaginous microorganisms rarely accumulate ω3-DPA (<5% of total lipid), strain T7 accumulated ω3-DPA with more than 20% of total fatty acids. The strain T7 was identified as a related species of Aurantiochytrium. In Aurantiochytrium sp. T7, ω3-DPA production reached 164mg/L culture broth, and the ω3-DPA content reached 23.5% of the total fatty acids when cultivated in a medium containing 2% glucose as the carbon source and 1% yeast extract as the nitrogen source, with a salinity equivalent to 50% of that of seawater and a pH in the acidic range (pH<5.5). Aurantiochytrium sp. T7 is a promising producer of high-purity ω3-DPA containing-lipid for the functional analysis of ω3-DPA whose physiological function has hardly been elucidated, and a useful strain for investigating the novel metabolic pathway of fatty acids.

Uncovering the Bioactivity of Aurantiochytrium sp.: a Comparison of Extraction Methodologies.

Aurantiochytrium sp. is an emerging alternative source of polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA), and squalene, playing an important role in the phasing out of traditional fish sources for these compounds. Novel lipid extraction techniques with a focus on sustainability and low environmental footprint are being developed for this organism, but the exploration of other added-value compounds within it is still very limited. In this work, a combination of novel green extraction techniques (high hydrostatic pressure extraction (HPE) and supercritical fluid extraction (SFE)) and traditional techniques (organic solvent Soxhlet extraction and hydrodistillation (HD)) was used to obtain lipophilic extracts of Aurantiochytrium sp., which were then screened for antioxidant (DPPH radical reduction capacity and ferric-reducing antioxidant potential (FRAP) assays), lipid oxidation protection, antimicrobial, anti-aging enzyme inhibition (collagenase, elastase and hyaluronidase), and anti-inflammatory (inhibition of NO production) activities. The screening revealed promising extracts in nearly all categories of biological activity tested, with only the enzymatic inhibition being low in all extracts. Powerful lipid oxidation protection and anti-inflammatory activity were observed in most SFE samples. Ethanolic HPEs inhibited both lipid oxidation reactions and microbial growth. The HD extract demonstrated high antioxidant, antimicrobial, and anti-inflammatory activities making, it a major contender for further studies aiming at the valorization of Aurantiochytrium sp. Taken together, this study presents compelling evidence of the bioactive potential of Aurantiochytrium sp. and encourages further exploration of its composition and application.

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

BackgroundTriacylglycerol (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.ResultsDiacylglycerol 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.ConclusionsThere 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.

Characterization of cellular development and fatty acid accumulation in thraustochytrid Aurantiochytrium strains of Taiwan

Abstract Long-chain saturated and polyunsaturated fatty acids of two new thraustochytrid isolates cultured from Taiwan mangroves, Aurantiochytrium sp. IMB169 and Aurantiochytrium sp. IMB171, were characterized through their cell growth and development in relation to their intracellular lipid accumulation using transmission electron microscopy. Flow cytometry in combination with the lipophilic fluorescent dye BODIPY 505/515 was used to stain and characterize intracellular lipid bodies in the two isolates. The transmission electron microscopy and flow cytometry analyses revealed a progressive accumulation of lipid products in IMB169 and IMB171. Further, selective BODIPY stained cells were successfully separated and enriched using flow cytometry at single cell level. Among the two isolates, IMB169 was found to produce a high level of docosahexaenoic acid. The qualitative and analytical results obtained using electron microscopy and flow cytometry studies were validated by gas chromatography (GC). In addition, a quantitative baseline was established using cell growth, flow cytometry and GC analyses for developing an efficient bioprocessing methodology to selectively enrich thraustochytrids phenotypes with desirable characteristics.

Single-Cell Oils from Oleaginous Microorganisms as Green Bio-Lubricants: Studies on Their Tribological Performance

Biolubricants refer to eco-friendly, biodegradable, and non-toxic lubricants. Their applications are still limited compared to mineral oils; however, their sustainable credentials are making them increasingly attractive. Vegetable oils are frequently used for this purpose. However, vegetable oils have issues of low lipid productivity, dependence on climatic conditions, and need for agricultural land. Microbial oils represent a more sustainable alternative. To ensure their widespread applicability, the suitability of microbial oils from a physicochemical point of view needs to be determined first. In this study, oils obtained from various oleagenic microbes—such as microalgae, thraustochytrids, and yeasts—were characterized in terms of their fatty acid profile, viscosity, friction coefficient, wear, and thermal stability. Oleaginous microalgal strains (Auxenochlorella protothecoides and Chlorella sorokiniana), thraustochytrids strains (Aurantiochytrium limacinum SR21 and Aurantiochytrium sp. T66), and yeast strains (Rhodosporidium toruloides and Cryptococcus curvatus) synthesized 64.5%, 35.15%, 47.89%, 47.93%, 56.42%, and 52.66% of lipid content, respectively. Oils from oleaginous microalgae (A. protothecoides and C. sorokiniana) and yeasts (R. toruloides and C. curvatus) possess excellent physicochemical and tribological qualities due to high amount of monounsaturated fatty acids (oleic acid C18:1 content, 56.38%, 58.82%, 46.67%, 38.81%) than those from oleaginous thraustochytrids (A. limacinum SR21 and Aurantiochytrium sp. T66; 0.96%, 0.08%, respectively) supporting their use as renewable and biodegradable alternatives to traditional mineral oil-based lubricants. Oil obtained from microalgae showed a lower friction coefficient than oils obtained from yeasts and thraustochytrids.

Aurantiochytrium sp. Meal Improved Body Fatty Acid Profile and Morphophysiology in Nile Tilapia Reared at Low Temperature

Aurantiochytrium sp. is a heterotrophic microorganism that produces docosahexaenoic acid (DHA), thus being considered as a possible replacement for fish oil in aquafeeds. We investigated the effect of Aurantiochytrium sp. meal (AM) dietary levels (0, 5, 10, 20, and 40 g kg−1) on Nile tilapia body and hepatopancreas fatty acid (FA) profile, body FA retention, somatic indices, and morphophysiological changes in the intestine and hepatopancreas, after feeding Nile tilapia juveniles (average initial weight 8.47 g) for 87 days at 22 °C. The 10AM diet was compared to a control diet containing cod liver oil (CLO), since their DHA concentration was similar. Within fish fed diets containing increasing levels of AM, there was a linear increase in n-3 FA content, especially DHA, which varied in the body (0.02 to 0.41 g 100 g−1) and hepatopancreas (0.15 to 1.05 g 100 g−1). The morphology of the intestines and hepatopancreas was positively affected in AM-fed fish. Fish fed 10AM showed less accumulation of n-3 FAs in the body and hepatopancreas when compared to fish fed CLO. Therefore, AM is an adequate substitute for fish oil in winter diets for Nile tilapia, with the supplementation of 40AM promoting the best results regarding intestine and hepatopancreas morphophysiology.

Evaluation of diverse biochemical stimulants to enhance growth, lipid and docosahexaenoic acid (DHA) production of Aurantiochytrium Sp. ATCC PRA-276

In the current investigation, the efficacy of five different biochemical stimulants; 2-phenylacetic acid (PAA), humic acid (HA), spermidine (SPD), 1-methylcyclopropene (1-MCP), and ferric chlorides (FC), as potential stimulant in augmenting the growth/biomass, lipid and docosahexaenoic acid (DHA) production of Aurantiochytrium sp. ATCC PRA-276 were elucidated. After 120 h of cultivation, the individual supplementation of PAA, HA, SPD, 1-MCP, and FC, elevates the biomass, total lipid and DHA production of Aurantiochytrium sp. ATCC PRA-276 by 32%–76%, 23%–34%, and 53%–86% in comparison to the control, respectively. The highest biomass and DHA productivities was observed with the supplementation of PAA and FC achieving 76% and 67% and 86% and 66%, compared to the control, respectively. PAA supplementation may have increased biomass production by reducing the lag period and extending the log phase. While only a marginal increment on biomass and DHA contents of Aurantiochytrium sp. ATCC PRA-276 was observed with the supplementation of 1-MCP. This study suggests the potential application of biochemical stimulants for improved growth, lipid and DHA productivities of Aurantiochytrium sp. ATCC PRA-276. • The effects of biochemical stimulants on Aurantiochytrium sp. ATCC PRA-276 growth, lipid, and DHA biosynthesis were studied. • Individual supplements of PAA, HA, and FC enhanced biomass, lipid, and DHA by 32%–76%, 23%–34%, and 53%–86%, respectively. • With the addition of PAA and FC, the maximum biomass and DHA productivities were achieved. • The addition of 1-MCP resulted in just a slight increase in the biomass and DHA contents of Aurantiochytrium sp. ATCC PRA-276.

Production of Docosahexaenoic Acid, DHA using Different Modes of Cultivation by Aurantiochytrium sp. SW1

Thraustochytrids, such as members of the genus Aurantiochytrium, are rich in docosahexaenoic acid (DHA, C22:6n-3) and represent a promising source of omega-3 fatty acids which plays a vital role in the enhancement of human health, particularly for neurological and visual functions. Different modes of cultivation (batch, fed-batch and repeated-batch) by Aurantiochytrium sp. SW1 were studied for effective docosahexaenoic acid (DHA) production. In this study, three different modes of fermentation were carried out in 1 L shake flasks with a working volume of 500 mL, incubated at 30 ºC and 200 rpm. Batch cultivation significantly exceeds the rest of cultivation modes, achieving maximal lipid and DHA concentrations of 11.22 g/L and 5.87 g/L, respectively, and DHA productivity of 0.061 g/L/h. Lipid and DHA concentration of the repeated-batch process decreased through the cycles for all three different types of replacement ratio (80, 90 and 95%). The average decrease percentage of DHA concentration for cycle one and cycle two were 21.76 and 32.52%, respectively. However, the fatty acid composition of lipids obtained in the cycles remained consistent with 16:0 and DHA being the most abundant fatty acids indicating that this mode of fermentation is highly useable for industrial applications.

A non-canonical \u03949-desaturase synthesizing palmitoleic acid identified in the thraustochytrid Aurantiochytrium sp. T66

Thraustochytrids are oleaginous marine eukaryotic microbes currently used to produce the essential omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3). To improve the production of this essential fatty acid by strain engineering, it is important to deeply understand how thraustochytrids synthesize fatty acids. While DHA is synthesized by a dedicated enzyme complex, other fatty acids are probably synthesized by the fatty acid synthase, followed by desaturases and elongases. Which unsaturated fatty acids are produced differs between different thraustochytrid genera and species; for example, Aurantiochytrium sp. T66, but not Aurantiochytrium limacinum SR21, synthesizes palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7). How strain T66 can produce these fatty acids has not been known, because BLAST analyses suggest that strain T66 does not encode any Δ9-desaturase-like enzyme. However, it does encode one Δ12-desaturase-like enzyme. In this study, the latter enzyme was expressed in A. limacinum SR21, and both C16:1 n-7 and C18:1 n-7 could be detected in the transgenic cells. Our results show that this desaturase, annotated T66Des9, is a Δ9-desaturase accepting C16:0 as a substrate. Phylogenetic studies indicate that the corresponding gene probably has evolved from a Δ12-desaturase-encoding gene. This possibility has not been reported earlier and is important to consider when one tries to deduce the potential a given organism has for producing unsaturated fatty acids based on its genome sequence alone.Key points• In thraustochytrids, automatic gene annotation does not always explain the fatty acids produced.• T66Des9 is shown to synthesize palmitoleic acid (C16:1 n-7).• T66des9 has probably evolved from Δ12-desaturase-encoding genes.

Docosahexaenoic Acid Is Naturally Concentrated at the sn-2 Position in Triacylglycerols of the Australian Thraustochytrid Aurantiochytrium sp. Strain TC 20.

The Labyrinthulomycetes or Labyrinthulea are a class of protists that produce a network of filaments that enable the cells to glide along and absorb nutrients. One of the main two Labyrinthulea groups is the thraustochytrids, which are becoming an increasingly recognised and commercially used alternate source of long-chain (LC, ≥C20) omega-3 containing oils. This study demonstrates, to our knowledge for the first time, the regiospecificity of the triacylglycerol (TAG) fraction derived from Australian thraustochytrid Aurantiochytrium sp. strain TC 20 obtained using 13C nuclear magnetic resonance spectroscopy (13C NMR) analysis. The DHA present in the TC 20 TAG fraction was determined to be concentrated in the sn-2 position, with TAG (16:0/22:6/16:0) identified as the main species present. The sn-2 preference is similar to that found in salmon and tuna oil, and differs to seal oil containing largely sn-1,3 LC-PUFA. A higher concentration of sn-2 DHA occurred in the thraustochytrid TC 20 oil compared to that of tuna oil.

Isolation and characterization of docosahexaenoic acid-producing novel strain Aurantiochytrium sp. ICTFD5: A sterol with vitamin d-cholecalciferol, and cellulase and lipase producing thraustochytrid

An oleaginous osmoheterotroph, thraustochytrid, is gaining attention due to its ability to produce high-value bioactive. In this study, the fast-growing thraustochytrid strain ICTFD5 was isolated, identified, and characterized for metabolites such as fatty acid profile, sterol profile, extracellular enzyme, biomass, and lipid production. The qualitative enzyme analysis of strain showed cellulase and lipase activity. The strain identified as Aurantiochytrium sp., and produced biomass (24.91 ± 0.5 g/L) accumulating high lipid (61.09% of dry cell weight) with simple fatty acid composition dominant with docosahexaenoic acid (45.24%) and palmitic acid (34.68%) in glucose-yeast extract medium at 25 °C for 96 h. The GCMS analysis showed cholecalciferol and ergosterol as vitamin D precursors and other sterols such as brassicasterol, lathosterol, ergosterol, chondrillasterol. Aurantiochytrium sp. ICTFD5 can be a potential commercial strain as a source of high-value nutraceutical omega 3 oil with DHA and vitamin d-cholecalciferol.

High-quality genome-scale metabolic model of Aurantiochytrium sp. T66.

The long-chain, ω-3 polyunsaturated fatty acids (PUFAs) (e.g., eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]), are essential for humans and animals, including marine fish species. Presently, the primary source of these PUFAs is fish oils. As the global production of fish oils appears to be reaching its limits, alternative sources of high-quality ω-3 PUFAs is paramount to support the growing aquaculture industry. Thraustochytrids are a group of heterotrophic protists with the capability to synthesize and accrue large amounts of DHA. Thus, the thraustochytrids are prime candidates to solve the increasing demand for ω-3 PUFAs using microbial cell factories. However, a systems-level understanding of their metabolic shift from cellular growth into lipid accumulation is, to a large extent, unclear. Here, we reconstructed a high-quality genome-scale metabolic model of the thraustochytrid Aurantiochytrium sp. T66 termed iVS1191. Through iterative rounds of model refinement and extensive manual curation, we significantly enhanced the metabolic scope and coverage of the reconstruction from that of previously published models, making considerable improvements with stoichiometric consistency, metabolic connectivity, and model annotations. We show that iVS1191 is highly consistent with experimental growth data, reproducing in vivo growth phenotypes as well as specific growth rates on minimal carbon media. The availability of iVS1191 provides a solid framework for further developing our understanding of T66's metabolic properties, as well as exploring metabolic engineering and process-optimization strategies in silico for increased ω-3 PUFA production.

Combined Metabolome and Lipidome Analyses for In-Depth Characterization of Lipid Accumulation in the DHA Producing Aurantiochytrium sp. T66.

Thraustochytrids are marine heterotrophic microorganisms known for their potential to accumulate docosahexaenoic acid (DHA)-enriched lipids. There have been many attempts to improve thraustochytrid DHA bioprocesses, especially through traditional optimization of cultivation and media conditions. Nevertheless, thraustochytrid-based bioprocesses are still not commercially competitive for high volume-low cost production of DHA. Thus, it is realized that genetic and metabolic engineering strategies are needed for the development of commercially competitive thraustochytrid DHA cell factories. Here, we present an analytical workflow for high resolution phenotyping at metabolite and lipid levels to generate deeper insight into the thraustochytrid physiology, with particular focus on central carbon and redox metabolism. We use time-series sampling during unlimited growth and nitrogen depleted triggering of DHA synthesis and lipid accumulation (LA) to show-case our methodology. The mass spectrometric absolute quantitative metabolite profiling covered glycolytic, pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) metabolites, amino acids, complete (deoxy)nucleoside phosphate pools, CoA and NAD metabolites, while semiquantitative high-resolution supercritical fluid chromatography MS/MS was applied for the lipid profiling. Interestingly, trace amounts of a triacylglycerols (TG) with DHA incorporated in all three acyl positions was detected, while TGs 16:0_16:0_22:6 and 16:0_22:6_22:6 were among the dominant lipid species. The metabolite profiling data indicated that lipid accumulation is not limited by availability of the acyl chain carbon precursor acetyl-CoA nor reducing power (NADPH) but rather points to the TG head group precursor glycerol-3-phosphate as the potential cause at the metabolite level for the gradual decline in lipid production throughout the cultivation. This high-resolution phenotyping provides new knowledge of changes in the central metabolism during growth and LA in thraustochytrids and will guide target selection for metabolic engineering needed for further improvements of this DHA cell factory.