Search Results (12)

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. Full article

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. Full article

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. Full article

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. Full article

We first sought to evaluate the effect of dietary supplementation with the docosahexaenoic acid (DHA)-rich microalgae, Aurantiochytrium limacinum (AURA), on rumen fermentation and the resistance of DHA to degradation and biohydrogenation by rumen microbes through ex vivo fermentation experiments. Subsequently, we sought to quantify the diet-derived DHA content of milk and the impact of AURA on microbial composition and metabolism in a pilot feeding trial with rumen-cannulated dairy cows. To achieve our aims, rumen fluid from cannulated cows was used as inoculum, and the effect of AURA inclusion on fermentation ex vivo was examined. At doses corresponding to the amount of AURA recommended for commercial production animals, only ~10% of DHA was degraded or biohydrogenated by rumen microorganisms. The results show that feeding with AURA had no effect on either total bacterial density or short-chain fatty acid production. Real-time quantitative PCR analysis of the rumen fluid samples collected during a seven-week in vivo trial revealed that microbes related to lactic acid metabolism and methanogenesis were significantly suppressed by the AURA-supplemented diet. The DHA concentration in milk increased over 25-fold with the AURA-supplemented diet and dropped by 30–40% within one week of washout. The addition of A. limacinum biomass to dairy cow diets resulted in positive effects on rumen microbial composition with no adverse effect on fermentation activity. AURA-derived DHA was stable, with only modest degradation in the rumen, and was successfully deposited in milk. This is the first study to investigate the effect of supplementing the diet of dairy cows with a protist-based biomass, namely, on important rumen fermentation parameters and on DHA deposition in milk, using a combination of ex vivo and in vivo approaches. Full article

Thraustochytrids are well-known unicellular heterotrophic marine protists because of their promising ability to accumulate docosahexaenoic acid (DHA). However, the implications of their unique genomic and metabolic features on DHA production remain poorly understood. Here, the effects of chemical and physical culture conditions on the cell mass and DHA production were investigated for a unique thraustochytrid strain, PKU#SW8, isolated from the seawater of Pearl River Estuary. All the tested fermentation parameters showed a significant influence on the cell mass and concentration and yield of DHA. The addition of monosaccharides (fructose, mannose, glucose, or galactose) or glycerol to the culture medium yielded much higher cell mass and DHA concentrations than that of disaccharides and starch. Similarly, organic nitrogen sources (peptone, yeast extract, tryptone, and sodium glutamate) proved to be beneficial in achieving a higher cell mass and DHA concentration. PKU#SW8 was found to grow and accumulate a considerable amount of DHA over wide ranges of KH₂PO₄ (0.125–1.0 g/L), salinity (0–140% seawater), pH (3–9), temperature (16–36 °C), and agitation (140–230 rpm). With the optimal culture conditions (glycerol, 20 g/L; peptone, 2.5 g/L; 80% seawater; pH 4.0; 28 °C; and 200 rpm) determined based on the shake-flask experiments, the cell mass and concentration and yield of DHA were improved up to 7.5 ± 0.05 g/L, 2.14 ± 0.03 g/L, and 282.9 ± 3.0 mg/g, respectively, on a 5-L scale fermentation. This study provides valuable information about the fermentation conditions of the PKU#SW8 strain and its unique physiological features, which could be beneficial for strain development and large-scale DHA production. Full article

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. Full article

Thraustochytrids are marine protists that naturally accumulate triacylglycerol with long chains of polyunsaturated fatty acids, such as ω3-docosahexaenoic acid (DHA). They represent a sustainable response to the increasing demand for these “essential” fatty acids (FAs). Following an attempt to transform a strain of Aurantiochytrium limacinum, we serendipitously isolated a clone that did not incorporate any recombinant DNA but contained two to three times more DHA than the original strain. Metabolic analyses indicated a deficit in FA catabolism. However, whole transcriptome analysis did not show down-regulation of genes involved in FA catabolism. Genome sequencing revealed extensive DNA deletion in one allele encoding a putative peroxisomal adenylate transporter. Phylogenetic analyses and yeast complementation experiments confirmed the gene as a peroxisomal adenylate nucleotide transporter (AlANT1), homologous to yeast ScANT1 and plant peroxisomal adenylate nucleotide carrier AtPNC genes. In yeast and plants, a deletion of the peroxisomal adenylate transporter inhibits FA breakdown and induces FA accumulation, a phenotype similar to that described here. In response to this metabolic event, several compensatory mechanisms were observed. In particular, genes involved in FA biosynthesis were upregulated, also contributing to the high FA accumulation. These results support AlANT1 as a promising target for enhancing DHA production in Thraustochytrids. Full article

The fatty acid composition of broiler chicken tissues can be increased by adding omega-3 rich ingredients to their diets. The purpose of this study was to compare the levels of tissue enrichment observed following the supplementation of broilers with the docosahexaenoic acid (DHA)-rich protist, Aurantiochytrium limacinum (AURA) for their whole life (42 days) or for the final 21-day fattening period. Day-old chicks (n = 350) were distributed among 35 pens (10 birds per pen) with each pen randomly assigned to one of five treatments: Control; 0.5% AURA from day 0–42; 1% AURA from day 0–42; 0.5% AURA from day 21–42; 1% AURA from day 21–42. Production parameters were recorded over the course of the study and the fatty acid profile of the breast, thigh, liver, kidney and skin with adhering fat was quantified at the end of the feeding period. The level of supplementation had a significant impact on the degree of omega-3 tissue enrichment, however, no differences were observed when the same dose was provided for 21 or 42 days. These results indicate that supplementation with AURA for a period of 21 days does not negatively affect broiler productivity and is the most efficient strategy to increase the nutritional value of broiler products. Full article

It is well established that the docosahexaenoic acid (DHA) content of commonly consumed meats, such as chicken, can be increased through dietary supplementation with DHA-rich ingredients. The purpose of this study was to investigate the tolerance of broilers to dietary supplementation with the unextracted biomass of a DHA-rich microalgae Aurantiochytrium limacinum, so as to ensure its safety, since it is accumulated in broiler meat. Healthy day-old male Ross 308 chicks (n = 1120) were evenly distributed to 32 pens (35 chicks per pen), with pens randomly allocated to one of four dietary treatments, each having eight replicates. The dietary groups included one untreated control and three treatments corresponding to three inclusion levels (0.5, 2.5, and 5.0%) of All-G-Rich^®, with the birds receiving the experimental diets ad libitum during the study (day 0–42). Bird survival, blood parameters, productivity, and breast and thigh DHA content were determined after 42 days of feeding. Supplementation at up to 10 times the intended use level had no negative effects on the mortality, blood parameters or productivity of the birds, while significant increases in the meat DHA content were observed. These results indicate that supplementation with Aurantiochytrium limacinum is a safe and effective way to increase broiler tissue DHA content. Full article

Aurantiochytrium limacinum, a marine heterotrophic protist/microalga has shown interesting yields of docosahexaenoic acid (DHA) when cultured with different carbon sources: glucose, pure and crude glycerol. A complete study in a lab-scale fermenter allowed for the characterization and comparison of the growth kinetic parameters corresponding to each carbon source. Artificial Marine Medium (AMM) with glucose, pure and crude glycerol offered similar biomass yields. The net growth rates (0.10–0.12 h⁻¹), biomass (0.7–0.8 g cells/g Substrate) and product (0.14–0.15 g DHA/g cells) yields, as well as DHA productivity were similar using the three carbon sources. Viable potential applications to valorize crude glycerol are envisioned to avoid an environmental problem due to the excess of byproduct. Full article

The Cre/loxP site-specific recombination system was applied to Aurantiochytrium limacinum to obtain a transformant without the antibiotic resistance marker gene. First, the enhanced green fluorescent protein gene (egfp) and chloramphenicol resistance gene (Cm^r), along with the two loxP loci, were integrated into the genome of A. limacinum OUC88 using 18S rDNA sequences as the homologous recombination sites. Then plasmid pSH65, containing a zeocin resistance gene (Ble^r) was transferred into A. limacinum OUC_CG. After induction with galactose, repeated passage in culture and PCR-based assessment, the pSH65 plasmid was lost and A. limacinum OUC_EG host was shown to no longer have resistance to 100 mg chloramphenicol/L or 5 mg zeocin/L. Through southern blotting and fluorescence detection, egfp was found to be integrated into the genome of A. limacinum OUC_EG, and EGFP was successfully expressed in the cells. The successful application of the Cre/loxP system demonstrates an experimental basis for genetic modification of A. limacinum so as to obtain transformed strains with no antibiotic resistance marker genes. Full article