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Nannochloropsis oceanica, a novel natural source of rumen-protected eicosapentaenoic acid (EPA) for ruminants.

ABSTRACT: We hypothesize that whole microalga biomass is a natural rumen-protected source of eicosapentaenoic acid (EPA, 20:5n-3) for ruminants. To test our hypothesis, we studied the ruminal biohydrogenation of EPA from two microalgae, Nannochloropsis oceanica and Phaeodactylum tricornutum using in vitro incubations with rumen fluid. A total mixed ration was incubated with: no EPA (control), EPA as free-fatty acid, N. oceanica spray-dried (SD), N. oceanica freeze-dried (FD), or P. tricornutum FD. The kinetics of EPA disappearance and of products formed during the 24?hours of incubation were evaluated, and complemented by deuterated-EPA incubation. Results showed that EPA metabolism from the N. oceanica was remarkably reduced compared with the P. tricornutum and free-EPA, and this reduction was even more effective with the N. oceanica FD. Our data also indicates that neither feed dry matter disappearance nor rumen microbial markers (branched-chain fatty acids and dimethyl acetals) were affected by EPA-sources. We reported for the first time the kinetics of EPA biohydrogenation class products and the unequivocal formation of 20:0 from EPA. Overall, N. oceanica shows a strong potential to be used as a natural dietary source of EPA to ruminants, nevertheless further studies are needed to verify its protection in vivo.

SUBMITTER: Alves SP

PROVIDER: S-EPMC6035222 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Nannochloropsis oceanica, a novel natural source of rumen-protected eicosapentaenoic acid (EPA) for ruminants.

Alves Susana P SP Mendonça Sofia H SH Silva Joana L JL Bessa Rui J B RJB

Scientific reports 20180706 1

We hypothesize that whole microalga biomass is a natural rumen-protected source of eicosapentaenoic acid (EPA, 20:5n-3) for ruminants. To test our hypothesis, we studied the ruminal biohydrogenation of EPA from two microalgae, Nannochloropsis oceanica and Phaeodactylum tricornutum using in vitro incubations with rumen fluid. A total mixed ration was incubated with: no EPA (control), EPA as free-fatty acid, N. oceanica spray-dried (SD), N. oceanica freeze-dried (FD), or P. tricornutum FD. The kin ...[more]

PMID: 29980726

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Project description:BackgroundAmong extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity.Methods and findingsA lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested.ConclusionsIn this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs.

Project description:BACKGROUND:Photosynthetic oleaginous microalgae are considered promising feedstocks for biofuels. The marine microalga, Nannochloropsis oceanica, has been attracting ever-increasing interest because of its fast growth, high triacylglycerol (TAG) content, and available genome sequence and genetic tools. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of TAG biosynthesis in the acyl-CoA-dependent pathway. Previous studies have identified 13 putative DGAT-encoding genes in the genome of N. oceanica, but the functional role of DGAT genes, especially type-I DGAT (DGAT1), remains ambiguous. RESULTS:Nannochloropsis oceanica IMET1 possesses two DGAT1 genes: NoDGAT1A and NoDGAT1B. Functional complementation demonstrated the capability of NoDGAT1A rather than NoDGAT1B to restore TAG synthesis in a TAG-deficient yeast strain. In vitro DGAT assays revealed that NoDGAT1A preferred saturated/monounsaturated acyl-CoAs and eukaryotic diacylglycerols (DAGs) for TAG synthesis, while NoDGAT1B had no detectable enzymatic activity. Assisted with green fluorescence protein (GFP) fusion, fluorescence microscopy analysis indicated the localization of NoDGAT1A in the chloroplast endoplasmic reticulum (cER) of N. oceanica. NoDGAT1A knockdown caused ~25% decline in TAG content upon nitrogen depletion, accompanied by the reduced C16:0, C18:0, and C18:1 in TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. NoDGAT1A overexpression, on the other hand, led to ~39% increase in TAG content upon nitrogen depletion, accompanied by the enhanced C16:0 and C18:1 in the TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. Interestingly, NoDGAT1A overexpression also promoted TAG accumulation (by ~2.4-fold) under nitrogen-replete conditions without compromising cell growth, and TAG yield of the overexpression line reached 0.49 g L-1 at the end of a 10-day batch culture, 47% greater than that of the control line. CONCLUSIONS:Taken together, our work demonstrates the functional role of NoDGAT1A and sheds light on the underlying mechanism for the biosynthesis of various TAG species in N. oceanica. NoDGAT1A resides likely in cER and prefers to transfer C16 and C18 saturated/monounsaturated fatty acids to eukaryotic DAGs for TAG assembly. This work also provides insights into the rational genetic engineering of microalgae by manipulating rate-limiting enzymes such as DGAT to modulate TAG biosynthesis and fatty acid composition for biofuel production.

Dataset Information

Nannochloropsis oceanica, a novel natural source of rumen-protected eicosapentaenoic acid (EPA) for ruminants.

Publications

Nannochloropsis oceanica, a novel natural source of rumen-protected eicosapentaenoic acid (EPA) for ruminants.

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