DHA Production Research Articles

Catalytic Potential-Guided Design of Multi-Enzymatic System for DHA Production from Glycerol

The growing demand for sustainable chemical production has spurred significant interest in biocatalysis. This study is framed within the biocatalytic production of 1,3-dihydroxyacetone (DHA) from glycerol, a byproduct of biodiesel manufacturing. The main goal of this study is to address the challenge of identifying the optimal operating conditions. To achieve this, catalytic potential, a lumped parameter that considers both the activity and stability of immobilized biocatalysts, was used to guide the design of a multi-enzymatic system. The multi-enzymatic system comprises glycerol dehydrogenase (GlyDH) and NADH oxidase (NOX). The enzymatic oxidation of glycerol to DHA catalyzed by GlyDH requires the cofactor NAD+. The integration of NOX into a one-pot reactor allows for the in situ regeneration of NAD+, enhancing the overall efficiency of the process. Furthermore, immobilization on Ni+2 agarose chelated supports, combined with post-immobilization modifications (glutaraldehyde crosslinking for GlyDH), significantly improved the stability and activity of both enzymes. The catalytic potential enabled the identification of the optimal operating conditions, which were 30 °C and pH 7.5, favoring NOX stability. This work establishes a framework for the rational design and optimization of multi-enzymatic systems. It highlights the crucial interplay between individual enzyme properties and process conditions to achieve efficient and sustainable biocatalytic transformations.

Microalgae Isolated from Singapore Mangrove Habitat as Promising Microorganisms for the Sustainable Production of Omega-3 Docosahexaenoic Acid

Docosahexaenoic acid (DHA, C22:6n-3) is an omega-3 fatty acid with beneficial effects for human health. In view of its increasing demand, DHA traditionally produced by marine fisheries will be insufficient, and an alternative sustainable source is urgently required. Here, we report the isolation and characterization of four novel microalgae strains, PLU-A, B, C and D, with a high DHA content of up to 45% from decayed mangrove samples collected from a coastal area in Singapore. Phylogenetic analysis revealed that these isolates were clustered with Schizochytrium sp. TK6 (OK244290.1) and were identified as Schizochytrium sp. strains. A medium optimization with Schizochytrium sp. PLU-D found a glucose-to-yeast extract ratio of 4:1 to be optimal for high biomass and lipid accumulation of up to 70% in shake flasks. In fed-batch fermentation scale-up with the Schizochytrium sp. PLU-D strain, this translates to 175 g/L dry biomass, 94 g/L lipid and 36.2 g/L DHA. Accordingly, the DHA titer obtained is superior to most of the scale-up production reported thus far, while the DHA content is comparable to two other commercially available DHA algae oils. These results suggest that Schizochytrium sp. PLU-D has high potential to be applied for the sustainable production of DHA.

Optimizing enzyme properties to enhance dihydroxyacetone production via methylglyoxal biosensor development

BackgroundDihydroxyacetone (DHA) stands as a crucial chemical material extensively utilized in the cosmetics industry. DHA production through the dephosphorylation of dihydroxyacetone phosphate, an intermediate product of the glycolysis pathway in Escherichia coli, presents a prospective alternative for industrial production. However, insights into the pivotal enzyme, dihydroxyacetone phosphate dephosphorylase (HdpA), remain limited for informed engineering. Consequently, the development of an efficient tool for high-throughput screening of HdpA hypermutants becomes imperative.ResultsThis study introduces a methylglyoxal biosensor, based on the formaldehyde-responding regulator FrmR, for the selection of HdpA. Initial modifications involved the insertion of the FrmR binding site upstream of the −35 region and into the spacer region between the −10 and −35 regions of the constitutive promoter J23110. Although the hybrid promoter retained constitutive expression, expression of FrmR led to complete repression. The addition of 350 μM methylglyoxal promptly alleviated FrmR inhibition, enhancing promoter activity by more than 40-fold. The methylglyoxal biosensor system exhibited a gradual increase in fluorescence intensity with methylglyoxal concentrations ranging from 10 to 500 μM. Notably, the biosensor system responded to methylglyoxal spontaneously converted from added DHA, facilitating the separation of DHA producing and non-producing strains through flow cytometry sorting. Subsequently, the methylglyoxal biosensor was successfully applied to screen a library of HdpA mutants, identifying two strains harboring specific mutants 267G > T and D110G/G151C that showed improved DHA production by 68% and 114%, respectively. Expressing of these two HdpA mutants directly in a DHA-producing strain also increased DHA production from 1.45 to 1.92 and 2.29 g/L, respectively, demonstrating the enhanced enzyme properties of the HdpA mutants.ConclusionsThe methylglyoxal biosensor offers a novel strategy for constructing genetically encoded biosensors and serves as a robust platform for indirectly determining DHA levels by responding to methylglyoxal. This property enables efficiently screening of HdpA hypermutants to enhance DHA production.

Production of Omega-3 Fatty Acid Concentrates from Common Kilka Oil: Optimization of the Urea Complexation Process.

There has been an increase in interest in the application of ω-3 PUFAs, especially EPA and DHA, in the development of various food products owing to their myriad health benefits. However, most fish oils do not contain more than 30% combined levels of EPA and DHA. In this study, through the urea complexation procedure, the production of EPA and DHA concentrate in their free fatty acids (FFAs) form was achieved from an enzymatic oil extracted from common kilka (Clupeonella cultriventris caspia). To gain the maximum value of EPA and DHA, the response surface methodology (RSM), which is an effective tool to categorize the level of independent variables onto the responses of an experimental process, was also used. Different variables including the urea-fatty acids (FAs) ratio (in the range of 2-6, w/w), the temperature of crystallization (in the range of -24-8 °C), and the time of crystallization (in the range of 8-40 h) were investigated by response surface methodology (RSM) for maximizing the EPA and DHA contents. Following the model validation, the levels of the variables at which the maximum desirability function (0.907 score) was obtained for response variables were 5:1 (urea-FAs ratio), -9 °C (the temperature of crystallization), and 24 h (the time of crystallization). Under these optimal conditions, increases of 2.2 and 4.4 times in the EPA and DHA concentrations were observed, respectively, and an increase in the concentrations of EPA and DHA from 5.39 and 13.32% in the crude oil to 12.07 and 58.36% in the ω-3 PUFA concentrates were observed, respectively. These findings indicate that the urea complexation process is efficient at optimizated conditions.

Continuous catalytic production of 1,3-dihydroxyacetone: Sustainable approach combining perfusion cultures and immobilized cells

Currently, the predominant method for the industrial production of 1,3-dihydroxyacetone (DHA) from glycerol involves fed-batch fermentation. However, previous research has revealed that in the biocatalytic synthesis of DHA from glycerol, when the DHA concentration exceeded 50 g·L−1, it significantly inhibited microbial growth and metabolism, posing a challenge in maintaining prolonged and efficient catalytic production of DHA. In this study, a new integrated continuous production and synchronous separation (ICSS) system was constructed using hollow fiber columns and perfusion culture technology. Additionally, a cell reactivation technique was implemented to extend the biocatalytic ability of cells. Compared with fed-batch fermentation, the ICSS system operated for 360 h, yielding a total DHA of 1237.8 ± 15.8 g. The glycerol conversion rate reached 97.7 %, with a productivity of 3.44 g·L−1·h−1, representing 485.0 % increase in DHA production. ICSS system exhibited strong operational characteristics and excellent performance, indicating significant potential for applications in industrial bioprocesses.

Production of docosahexaenoic acid by a novel isolated Aurantiochytrium sp. 6-2 using fermented defatted soybean as a nitrogen source for sustainable fish feed development.

We focused on the production of docosahexaenoic acid (DHA)-containing microbial lipids by Aurantiochytrium sp. using of defatted soybean (DS) as a nitrogen source. Defatted soybean is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytrium sp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytrium sp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytrium sp. 6-2 showed 15.8±3.4g/L DHA productivity (in 54.8±12.1g/L total fatty acid production) in 1L of the FDS medium. Therefore, DHA produced by Aurantiochytrium sp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.

Integration analysis of ATAC-seq and RNA-seq provides insight into fatty acid biosynthesis in Schizochytrium limacinum under nitrogen limitation stress

BackgroundSchizochytrium limacinum holds significant value utilized in the industrial-scale synthesis of natural DHA. Nitrogen-limited treatment can effectively increase the content of fatty acids and DHA, but there is currently no research on chromatin accessibility during the process of transcript regulation. The objective of this research was to delve into the workings of fatty acid production in S. limacinum by examining the accessibility of promoters and profiling gene expressions.ResultsResults showed that differentially accessible chromatin regions (DARs)-associated genes were enriched in fatty acid metabolism, signal transduction mechanisms, and energy production. By identifying and annotating DARs-associated motifs, the study obtained 54 target transcription factor classes, including BPC, RAMOSA1, SPI1, MYC, and MYB families. Transcriptomics results revealed that several differentially expressed genes (DEGs), including SlFAD2, SlALDH, SlCAS1, SlNSDHL, and SlDGKI, are directly related to the biosynthesis of fatty acids, meanwhile, SlRPS6KA, SlCAMK1, SlMYB3R1, and SlMYB3R5 serve as transcription factors that could potentially influence the regulation of fatty acid production. In the integration analysis of DARs and ATAC-seq, 13 genes were identified, which were shared by both DEGs and DARs-associated genes, including SlCAKM, SlRP2, SlSHOC2, SlTN, SlSGK2, SlHMP, SlOGT, SlclpB, and SlDNAAF3.ConclusionsSlCAKM may act as a negative regulator of fatty acid and DHA synthesis, while SlSGK2 may act as a positive regulator, which requires further study in the future. These insights enhance our comprehension of the processes underlying fatty acid and DHA production in S. limacinum. They also supply a foundational theoretical framework and practical assistance for the development of strains rich in fatty acids and DHA.

Recycling Fermentation Strategy for Waste Cellular Residues in the Production of Polyunsaturated Fatty Acids

Cellular residues after lipid extraction are the primary waste by-products of microbial polyunsaturated fatty acid production. To mitigate the discharge of cellular residues and reduce the cost of DHA and ARA production, this study examined the utilization of enzymatically hydrolyzed Aurantiochytrium cellular residues (ACRs) and Mortierella alpina cellular residues (MCRs) as nitrogen sources. Results demonstrated that ACRs and MCRs could partially substitute yeast extract (YE) without adverse effects on DHA and ARA fermentation. Moreover, the implementation of a new fermentation medium incorporating ACRs, MCRs, and YE as mixed nitrogen sources resulted in DHA and ARA yields of 17.78 and 5.77 g/L, respectively. These values represented increases of 10.37% and 9.28% compared to traditional cultural methods, while simultaneously reducing the usage of YE by 80% and 60%, respectively. Therefore, the strategy of recycling waste cellular residues presents a novel approach for reducing the costs and environmental impact associated with microbial fermentations.

Fine-tuning of key parameters to enhance biomass and nutritional polyunsaturated fatty acids production from Thraustochytrium sp.

The escalating demand for long-chain polyunsaturated fatty acids (PUFAs) due to their vital health effects has deepened the exploration of sustainable sources. Thraustochytrium sp. stands out as a promising platform for omega-3 and 6 PUFA production. This research strategically optimizes key parameters: temperature, salinity, pH, and G:Y:P ratio and the optimized conditions for maximum biomass, total lipid, and DHA enhancement were 28 °C, 50 %, 6, and 10:1:2 respectively. Process optimization enhanced 32.30 and 31.92 % biomass (9.88 g/L) and lipid (6.57 g/L) yield. Notably, DHA concentration experienced a substantial rise of 69.91 % (1.63 g/L), accompanied by notable increases in EPA and DPA by 82.69 % and 31.47 %, respectively. MANOVA analysis underscored the statistical significance of the optimization process (p < 0.01), with all environmental factors significantly influencing biomass and lipid data (p < 0.05), particularly impacting DHA production. Thraustochytrium sp. can be a potential source of commercial DHA production with the fine-tuning of these key process parameters.

Electroporation-induced mutation and transcriptome analysis for high DHA production in Schizochytrium limacinum GCD2032

The development of the DHA industry requires continuous iterative upgrading of high-yield DHA-producing strains. In this study, a DHA high-producing strain named S. limacinum GCD2032 was screened through electroporation using zeocin as the selective agent and S. limacinum BCD as the wild strain. The production enhancement mechanism was elucidated through comparative transcriptome studies between S. limacinum GCD2032 and the wild strain. The fatty acid content, DHA content and DHA yield of S. limacinum GCD2032 were 3.51 %, 95.51 % and 71.67 % higher than those of S. limacinum BCD, respectively. Transcriptome analysis indicated that genes involved in the aerobic oxidation of glucose, the pentose phosphate pathway, the citric acid pyruvate cycle, and the PKS pathway for DHA production were all upregulated and genes involved in β-oxidation in peroxisomes were downregulated, resulting in the enhanced DHA content of S. limacinum GCD2032. This study provides an efficient iterative upgrading method and key candidate genes for DHA production in Schizochytrium. S. limacinum GCD2032 is a promising strain for further industrialization.

Isolation and Characterization of Thraustochytrium Trk-23 Producing Docosahexaenoic Acid from North Kalimantan, Indonesia.

&lt;b&gt;Background and Objective:&lt;/b&gt; The mangrove forest located in Tarakan Bay, North Kalimantan Province, Indonesia is geographically distant from human settlement and industrial activities. Thus, it remains unaffected by the presence of human-generated waste and industrial pollutants. This study aims to isolate and characterize the microalgae Thraustochytrids from this location, which can produce docosahexaenoic acid (DHA, C22:6, n-3). It is anticipated that these microalgae possess the potential for commercial production of DHA. &lt;b&gt;Materials and Methods:&lt;/b&gt; The fallen leaf sample was collected from the mangrove forest, then isolated and purified by scratching technique until a single colony state. The pure isolate then be identified by 18S rDNA. The sequences were then analyzed for similarities using the Basic Local Alignment Search Tool (BLAST). The phylogenetic trees were carried out using the MEGA 6 program. The choice of phylogenetic trees was based on maximum likelihood. &lt;b&gt;Results:&lt;/b&gt; The identification 18S rDNA gene, a strain namely Trk-23, was identified as &lt;i&gt;Thraustochytrium&lt;/i&gt; sp. In the optimization of the cell growth of this strain, it was found that &lt;i&gt;Thraustochytrium&lt;/i&gt; Trk-23 has maximum growth at 6.0% glucose, 1.0% yeast extract and 50% natural seawater, at a pH of 5.0 and a temperature of 30°C. The maximum lipid content is 6.0 g and the DHA proportion is 41.6% of the total fatty acid content with a DHA yield is 2.5 g L&lt;sup&gt;1&lt;/sup&gt;. &lt;b&gt;Conclusion:&lt;/b&gt; Some places in North Kalimantan are still free from industrial pollution and rich with &lt;i&gt;Thraustochytrium&lt;/i&gt; sp., which is why it can find &lt;i&gt;Thraustochytrium&lt;/i&gt; Trk-23. Due to its potency, &lt;i&gt;Thraustochytrium&lt;/i&gt; Trk-23 is the promising candidate microalgae strain for producing DHA commercially.

Multi-omics view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid catabolism increases polyketide-synthase-driven docosahexaenoic production to high selectivity at the gram scale

DHA is a marine PUFA of commercial value, given its multiple health benefits. The worldwide emerging shortage in DHA supply has increased interest in microbial cell factories that can provide the compound de novo. In this regard, the present work aimed to improve DHA production in the oleaginous yeast strain Y. lipolytica Af4, which synthetized the PUFA via a heterologous myxobacterial polyketide synthase (PKS)-like gene cluster. As starting point, we used transcriptomics, metabolomics, and 13C-based metabolic pathway profiling to study the cellular dynamics of Y. lipolytica Af4. The shift from the growth to the stationary DHA-production phase was associated with fundamental changes in carbon core metabolism, including a strong upregulation of the PUFA gene cluster, as well as an increase in citrate and fatty acid degradation. At the same time, the intracellular levels of the two DHA precursors acetyl-CoA and malonyl-CoA dropped by up to 98% into the picomolar range. Interestingly, the degradation pathways for the ketogenic amino acids l-lysine, l-leucine, and l-isoleucine were transcriptionally activated, presumably to provide extra acetyl-CoA. Supplementation with small amounts of these amino acids at the beginning of the DHA production phase beneficially increased the intracellular CoA-ester pools and boosted the DHA titer by almost 40%. Isotopic 13C-tracer studies revealed that the supplements were efficiently directed toward intracellular CoA-esters and DHA. Hereby, l-lysine was found to be most efficient, as it enabled long-term activation, due to storage within the vacuole and continuous breakdown. The novel strategy enabled DHA production in Y. lipolytica at the gram scale for the first time. DHA was produced at a high selectivity (27% of total fatty acids) and free of the structurally similar PUFA DPA, which facilitates purification for high-value medical applications that require API-grade DHA. The assembled multi-omics picture of the central metabolism of Y. lipolytica provides valuable insights into this important yeast. Beyond our work, the enhanced catabolism of ketogenic amino acids seems promising for the overproduction of other compounds in Y. lipolytica, whose synthesis is limited by the availability of CoA ester precursors.

Eutrophication effect on production and transfer of omega-3 fatty acids in boreal lake food webs

Eutrophication, i.e. increasing level of nutrients and primary production, is a central environmental change of lakes globally with wide effects on food webs. However, how eutrophication affects the synthesis of physiologically essential biomolecules (omega-3 fatty acids) and their transfer to higher trophic levels at the whole food web level is not well understood. We assessed food web (phytoplankton, zooplankton, and fish) biomass, community structure and fatty acid content (eicosapentaenoic acid [EPA], and docosahexaenoic acid [DHA]), together with fatty acid specific primary production in 12 Finnish boreal lakes covering the total nutrient gradient from oligotrophic to highly eutrophic lakes (4–140 μg TP l−1; 413–1814 μg TN l−1). Production was measured as the incorporation of 13C-NaHCO3 into phytoplankton fatty acids and differentiated into volumetric production (production per litre of water) and productivity (production per phytoplankton biomass). Increases in nutrients led to higher biomass of phytoplankton, zooplankton and fish communities while also affecting community composition. Eutrophication negatively influenced the contribution of phytoplankton biomass preferentially grazed by zooplankton (<35 μm). Total volumetric production saturated at high phytoplankton biomass while EPA volumetric production presented a logarithmic relationship with nutrient increase. Meanwhile, total and EPA productivity had unimodal responses to this change in nutrients. DHA volumetric production and productivity presented large variation with increases in total phosphorus, but a unimodal model best described DHA changes with eutrophication. Results showed that eutrophication impaired the transfer of EPA and DHA into zooplankton and fish, showing a clear negative impact in some species (e.g. perch) while having no effect in other species (e.g. roach, ruffe). Results show non-linear trends in fatty acid production and productivity peaking at nutrient concentrations 22–35 μg l−1 TP followed by a gradual decrease.

355-OR: Targeting a Lipid Droplet Hydrolase-Mediated Lipid Signaling for Treatment of Type 2 Diabetes

Lipid droplets (LDs) are cytosolic organelles that serve as the major energy reservoir in adipocytes. Excessive accumulation of neutral lipids in LDs has been linked to obesity-associated type 2 diabetes. The dynamics of LDs is regulated by multiple proteins bound to their surfaces. Recently, carboxylesterase 1d (Ces1d) was identified as a LD-targeting lipid hydrolase. It has been showed that blockage of Ces1d in adipose tissue led to not only decreased lipolysis, but also impaired β-adrenergic signaling-stimulated thermogenesis. The goal of the current study is to define the mechanism governing the function of Ces1d on lipid metabolism. The hypothesis is that the LD-targeting Ces1d generates free fatty acids (FFA) “third messenger” that trigger specific lipid signaling in adipose tissue which further affect the whole-body metabolic homeostasis. To test this hypothesis, an adipose tissue specific Ces1d knockout mouse model (Adipo-Ces1d KO) was generated. Mass Spectrometry analysis revealed that circulating docosahexaenoic acid (DHA, C22:6 n=3), an established PPARγ ligand, was dramatically decreased in Adipo-Ces1d KO mice. The production of DHA was confirmed by an in vitro lipid digestion assay on the recombinant Ces1d. Lack of DHA signaling further led to decreased transactivation of PPARγ in adipose tissue of the Adipo-Ces1d KO mice. As a result, the expression of thermogenic gene, UCP1 was significantly downregulated. The result agrees with previous observation that Adipo-Ces1d KO mice lost their ability to defense body temperature when exposed to coldness. More interestingly, the Adipo-Ces1d KO mice gained more body weight with increased fat mass during a HFD challenge, suggesting they experienced decreased energy expenditure in the whole process. The findings thus highlight the key role of a novel lipid signaling pathway mediated by Ces1d in energy expenditure and hence pinpoint it as a potential target for treatment of obesity-induced type 2 diabetes. Disclosure X.Li: None. K.Sun: None. Funding National Institutes of Health (R56DK124419, R01DK129815)

Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase is essential for the production of DHA in zebrafish

Compared with other species, freshwater fish are more capable of synthesizing DHA via same biosynthetic pathways. Freshwater fish have a “Sprecher” pathway to biosynthesize DHA in a peroxisome-dependent manner. Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) is involved in the hydration and dehydrogenation reactions of fatty acid β-oxidation in peroxisomes. However, the role of Ehhadh in the synthesis of DHA in freshwater fish remains largely unclear. In this study, the knockout of Ehhadh significantly inhibited DHA synthesis in zebrafish. Liver transcriptome analysis showed that Ehhadh deletion significantly inhibited SREBF and PPAR signaling pathways and decreased the expression of PUFA synthesis-related genes. Our results from the analysis of transgenic zebrafish (Tg:Ehhadh) showed that Ehhadh overexpression significantly increased the DHA content in the liver and significantly upregulated the expression of genes related to PUFA synthesis. In addition, the DHA content in the liver of Tg:Ehhadh fed with linseed oil was significantly higher than that of wildtype, but the expression of PUFA synthesis-related genes fads2 and elovl2 were significantly lower, indicating that Ehhadh had a direct effect on DHA synthesis. In conclusion, our results showed that Ehhadh was essential for DHA synthesis in the “Sprecher” pathway, and Ehhadh overexpression could promote DHA synthesis. This study provides insight into the role of Ehhadh in freshwater fish.

Simultaneous photoautotrophic production of DHA and EPA by Tisochrysis lutea and Microchloropsis salina in co-culture

Marine microalgae have received much attention as a sustainable source of the two health beneficial omega-3-fatty acids docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5). However, photoautotrophic monocultures of microalgae can only produce either DHA or EPA enriched biomass. An alternative may be the photoautotrophic co-cultivation of Tisochrysis lutea as DHA-producer with Microchloropsis salina for simultaneous EPA production to obtain EPA- and DHA-rich microalgae biomass in a nutritionally balanced ratio. Photoautotrophic co-cultivation processes of T. lutea and M. salina were studied, applying scalable and fully controlled lab-scale gas-lift flat-plate photobioreactors with LED illumination for dynamic climate simulation of a repeated sunny summer day in Australia [day–night cycles of incident light (PAR) and temperature]. Monocultures of both marine microalgae were used as reference batch processes. Differences in the autofluorescence of both microalgae enabled the individual measurement, of cell distributions in co-culture, by flow cytometry. The co-cultivation of T. lutea and M. salina in artificial sea water with an inoculation ratio of 1:3 resulted in a balanced biomass production of both microalgae simultaneously with a DHA:EPA ratio of almost 1:1 (26 mgDHA gCDW−1, and 23 mgEPA gCDW−1, respectively) at harvest after depletion of the initially added fertilizer. Surprisingly, more microalgae biomass was produced within 8 days in co-cultivation with an increase in the cell dry weight (CDW) concentration by 31%, compared to the monocultures with the same amount of light and fertilizer. What is more, DHA-content of the microalgae biomass was enhanced by 33% in the co-culture, whereas EPA-content remained unchanged compared to the monocultures.Graphical

Efficient 1,3-dihydroxyacetone biosynthesis in Gluconobacter oxydans using metabolic engineering and a fed-batch strategy

1,3-Dihydroxyacetone (DHA) is a commercially important chemical and widely used in cosmetics, pharmaceuticals, and food industries as it prevents excessive water evaporation, and provides anti-ultraviolet radiation protection and antioxidant activity. Currently, the industrial production of DHA is based on a biotechnological synthetic route using Gluconobacter oxydans. However, achieving higher production requires more improvements in the synthetic process. In this study, we compared DHA synthesis levels in five industrial wild-type Gluconobacter strains, after which the G. oxydans WSH-003 strain was selected. Then, 16 dehydrogenase genes, unrelated to DHA synthesis, were individually knocked out, with one strain significantly enhancing DHA production, reaching 89.49 g L−1 and 42.27% higher than the wild-type strain. By optimizing the culture media, including seed culture and fermentation media, DHA production was further enhanced. Finally, using an established fed-batch fermentation system, DHA production reached 198.81 g L−1 in a 5 L bioreactor, with a glycerol conversion rate of 82.84%.Graphical

Mendelian Randomization Indicates a Causal Role for Omega-3 Fatty Acids in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal system. Omega-3 (ω3) fatty acids are polyunsaturated fatty acids (PUFAs) that are largely obtained from diet and have been speculated to decrease the inflammatory response that is involved in IBD; however, the causality of this association has not been established. A two-sample Mendelian randomization (MR) was used to assess genetic associations between 249 circulating metabolites measured in the UK Biobank as exposures and IBD as the outcome. The genome-wide association study summary level data for metabolite measurements and IBD were derived from large European ancestry cohorts. We observed ω3 fatty acids as a significant protective association with IBD, with multiple modes of MR evidence replicated in three IBD summary genetic datasets. The instrumental variables that were involved in the causal association of ω3 fatty acids with IBD highlighted an intronic SNP, rs174564, in FADS2, a protein engaged in the first step of alpha-linolenic acid desaturation leading to anti-inflammatory EPA and thence DHA production. A low ratio of ω3 to ω6 fatty acids was observed to be a causal risk factor, particularly for Crohn's disease. ω3 fatty acid supplementation may provide anti-inflammatory responses that are required to attenuate inflammation that is involved in IBD.

S3.4e Unraveling the role of DOG genes in a novel alternative pathway of glycerol biosynthesis in Candida albicans and its influence on virulence

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMDOG genes, encoding for 2-deoxyglucose-6-phosphate phosphatase are low molecular weight phosphatases, with an unknown biological function. In contrast to Saccharomyces cerevisiae which has two DOG homologs, C. albicans only has one DOG gene. We hypothesized that DOG1 plays an important role under osmotic or ionic stress by biosynthesizing glycerol, which is known to be vital for biofilm formation and virulence of this pathogenic yeast, via a novel alternative pathway. The known classical pathway of glycerol production begins when the glycolytic intermediate molecule dihydroxyacetone phosphate (DHAP) is converted into glycerol-3-phosphate (G-3-P) by a pair of glycerol-3-phosphate-dehydrogenases, Gpd1 and Gpd2. G-3-P is further dephosphorylated into glycerol by glycerol-3-phosphate-phosphatases, Gpp1 and Gpp2. However, an alternative pathway, where DHAP is dephosphorylated into DHA, which is subsequently converted into glycerol has been proposed, but the enzymes involved in this process have not yet been described. We recently showed that in Saccharomyces cerevisiae, the DOG enzymes are involved in the production of DHA from DHAP, thereby allowing the synthesis of glycerol in the absence of the classical pathway. Overexpression of the DOG genes restored the osmo-tolerance of the gpp1Δ gpp2Δand gpd1Δ gpd2Δ double deletion strains. Furthermore, purified DOG enzymes could also convert DHAP into DHA (Awasthy et al., submitted). Since DOG1 has a potential role in biosynthesizing glycerol via an unconventional route, we are interested to determine its contribution in influencing virulence and biofilm formation in Candida albicans. This putative pathway has been overlooked for the past two decades, leaving behind an evident knowledge gap. We have now generated multiple deletion strains, using CRISPR Cas9, for the C. albicans counterparts of the GPP, GPD, and DOG genes as well as multiple DOG1 overexpression strains in which we observed the restoration of osmotic stress tolerance phenotypically and via growth curves. We also have NMR data showing the accumulation of various metabolites of central metabolism in these strains. Additionally, we have determined the possible role of DOG1 on virulence factors like morphogenesis, adhesion, and biofilm formation in vitro as well as in vivo, the latter with our catheter-based biofilm subcutaneous mouse model system. We also linked DOG1 and its role in glycerol biosynthesis to the survival of C. albicans inside macrophages. Finally, we would be setting up a high throughput small compound screening for this phosphatase as a potential antifungal drug target.

Nrf2a dependent and independent effects of early life exposure to 3,3'-dichlorobiphenyl (PCB-11) in zebrafish (Danio rerio).

The environmental pollutant 3,3'-dichlorobiphenyl (PCB-11) is a lower-chlorinated polychlorinated biphenyl (PCB) congener present in air and water samples. Both PCB-11 and its metabolite, 4-PCB-11-Sulfate, are detected in humans, including in pregnant women. Previous research in zebrafish (Danio rerio) has shown that 0.2μM exposures to 4-PCB-11-Sulfate starting at 1 day post fertilization (dpf) increase hepatic neutral lipid accumulation in larvae at 15 dpf. Here, we explored whether nuclear factor erythroid 2-related factor 2 (Nrf2), known as the master-regulator of the adaptive response to oxidative stress, contributes to metabolic impacts of 4-PCB-11-Sulfate. For this work, embryos were collected from homozygous wildtype or Nrf2a mutant adult zebrafish that also express GFP in pancreatic β-cells, rendering Tg(ins:GFP;nrf2afh318+/+) and Tg(ins:GFP;nrf2afh318-/-) lines. Exposures were conducted from 1-15 dpf to either 0.05% DMSO or DMSO-matched 0.2µM 4-PCB-11-Sulfate, and at 15 dpf subsets of larvae were imaged for overall morphology, primary pancreatic islet area, and collected for fatty acid profiling and RNAseq. At 15 dpf, independent of genotype, fish exposed to 4-PCB-11-Sulfate survived significantly more at 80-85% compared to 65-73% survival for unexposed fish, and had primary pancreatic islets 8% larger compared to unexposed fish. Fish growth at 15 dpf was dependent on genotype, with Nrf2a mutant fish a significant 3-5% shorter than wildtype fish, and an interaction effect was observed where Nrf2a mutant fish exposed to 4-PCB-11-Sulfate experienced a significant 29% decrease in the omega-3 fatty acid DHA compared to unexposed mutant fish. RNAseq revealed 308 differentially expressed genes, most of which were dependent on genotype. These findings suggest that Nrf2a plays an important role in growth as well as for DHA production in the presence of 4-PCB-11-Sulfate. Further research would be beneficial to understand the importance of Nrf2a throughout the lifecourse, especially in the context of toxicant exposures.