Microbial and genetically engineered oils as replacements for fish oil in aquaculture feeds.

As the global population grows more of our fish and seafood are being farmed. Fish are the main dietary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, but these cannot be produced in sufficient quantities as are now required for human health. Farmed fish have traditionally been fed a diet consisting of fishmeal and fish oil, rich in n-3 LC-PUFA. However, the increase in global aquaculture production has resulted in these finite and limited marine ingredients being replaced with sustainable alternatives of terrestrial origin that are devoid of n-3 LC-PUFA. Consequently, the nutritional value of the final product has been partially compromised with EPA and DHA levels both falling. Recent calls from the salmon industry for new sources of n-3 LC-PUFA have received significant commercial interest. Thus, this review explores the technologies being applied to produce de novo n-3 LC-PUFA sources, namely microalgae and genetically engineered oilseed crops, and how they may be used in aquafeeds to ensure that farmed fish remain a healthy component of the human diet.

LC-PUFA-enriched oil production by microalgae: accumulation of lipid and triacylglycerols containing n-3 LC-PUFA is triggered by nitrogen limitation and inorganic carbon availability in the marine haptophyte Pavlova lutheri.

In most microalgal species, triacyglycerols (TAG) contain mostly saturated and monounsaturated fatty acids, rather than PUFA, while PUFA-enriched oil is the form most desirable for dietary intake. The ability of some species to produce LC-PUFA-enriched oil is currently of specific interest. In this work, we investigated the role of sodium bicarbonate availability on lipid accumulation and n-3 LC-PUFA partitioning into TAG during batch cultivation of Pavlova lutheri. Maximum growth and nitrate uptake exhibit an optimum concentration and threshold tolerance to bicarbonate addition (~9 mM) above which both parameters decreased. Nonetheless, the transient highest cellular lipid and TAG contents were obtained at 18 mM bicarbonate, immediately after combined alkaline pH stress and nitrate depletion (day nine), while oil body and TAG accumulation were highly repressed with low carbon supply (2 mM). Despite decreases in the proportions of EPA and DHA, maximum volumetric and cellular EPA and DHA contents were obtained at this stage due to accumulation of TAG containing EPA/DHA. TAG accounted for 74% of the total fatty acid per cell, containing 55% and 67% of the overall cellular EPA and DHA contents, respectively. These results clearly demonstrate that inorganic carbon availability and elevated pH represent two limiting factors for lipid and TAG accumulation, as well as n-3 LC-PUFA partitioning into TAG, under nutrient-depleted P. lutheri cultures.

Metabolic engineering plant seeds with fish oil-like levels of DHA.

BACKGROUND: Omega-3 long-chain (≥C(20)) polyunsaturated fatty acids (ω3 LC-PUFA) have critical roles in human health and development with studies indicating that deficiencies in these fatty acids can increase the risk or severity of cardiovascular and inflammatory diseases in particular. These fatty acids are predominantly sourced from fish and algal oils, but it is widely recognised that there is an urgent need for an alternative and sustainable source of EPA and DHA. Since the earliest demonstrations of ω3 LC-PUFA engineering there has been good progress in engineering the C(20) EPA with seed fatty acid levels similar to that observed in bulk fish oil (∼18%), although undesirable ω6 PUFA levels have also remained high. METHODOLOGY/PRINCIPAL FINDINGS: The transgenic seed production of the particularly important C(22) DHA has been problematic with many attempts resulting in the accumulation of EPA/DPA, but only a few percent of DHA. This study describes the production of up to 15% of the C(22) fatty acid DHA in Arabidopsis thaliana seed oil with a high ω3/ω6 ratio. This was achieved using a transgenic pathway to increase the C(18) ALA which was then converted to DHA by a microalgal Δ6-desaturase pathway. CONCLUSIONS/SIGNIFICANCE: The amount of DHA described in this study exceeds the 12% level at which DHA is generally found in bulk fish oil. This is a breakthrough in the development of sustainable alternative sources of DHA as this technology should be applicable in oilseed crops. One hectare of a Brassica napus crop containing 12% DHA in seed oil would produce as much DHA as approximately 10,000 fish.

Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants.

The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes. However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation.

The production of unusual fatty acids in transgenic plants.

The ability to genetically engineer plants has facilitated the generation of oilseeds synthesizing non-native fatty acids. Two particular classes of fatty acids are considered in this review. First, so-called industrial fatty acids, which usually contain functional groups such as hydroxyl, epoxy, or acetylenic bonds, and second, very long chain polyunsaturated fatty acids normally found in fish oils and marine microorganisms. For industrial fatty acids, there has been limited progress toward obtaining high-level accumulation of these products in transgenic plants. For very long chain polyunsaturated fatty acids, although they have a much more complex biosynthesis, accumulation of some target fatty acids has been remarkably successful. In this review, we consider the probable factors responsible for these different outcomes, as well as the potential for further optimization of the transgenic production of unusual fatty acids in transgenic plants.

Application of support vector machines to 1H NMR data of fish oils: methodology for the confirmation of wild and farmed salmon and their origins.

Support vector machines (SVMs) were used as a novel learning machine in the authentication of the origin of salmon. SVMs have the advantage of relying on a well-developed theory and have already proved to be successful in a number of practical applications. This paper provides a new and effective method for the discrimination between wild and farm salmon and eliminates the possibility of fraud through misrepresentation of the country of origin of salmon. The method requires a very simple sample preparation of the fish oils extracted from the white muscle of salmon samples. (1)H NMR spectroscopic analysis provides data that is very informative for analysing the fatty acid constituents of the fish oils. The SVM has been able to distinguish correctly between the wild and farmed salmon; however ca. 5% of the country of origins were misclassified.

Pyloric ceca are significant sites of newly synthesized 22:6n-3 in rainbow trout (Oncorhynchus mykiss).

In this pulse-chase study, rainbow trout fed a diet containing deuterated (D5) (17,17,18,18,18)-18:3n-3 ethyl ester accumulated D5-22:6n-3 in pyloric ceca to a greater extent than in liver 2 d post-dose. The ratio of newly synthesized D5-22:6n-3 in ceca to that in liver 2 d after feeding D5-18:3n-3 was 4.7 +/- 1.2 when expressed as per mg tissue and 5.2 +/- 2.4 when expressed as per mg protein. The amount of D5-22:6n-3 in ceca then declined whereas that in liver and blood increased, with the ratio of ceca to liver falling to 1.7 and 1.4, respectively, by day 5 and approaching unity by day 9. A crude cecal mucosa fraction contained 123 +/- 50 ng D5-22:6n-3/mg protein/mg D5-18:3n-3 eaten 2 d after feeding the tracer, compared with 35 +/- 21 ng D5-22:6n-3/mg protein/mg D5-18:3n-3 eaten in liver. Three days later the amount in cecal mucosa had fallen by one-third and that in liver had increased threefold. Most of the D5-18:3n-3 was catabolized very rapidly. The ratio of D5-18:3n-3 to 21:4n-6 (a relatively inert FA marker) in the diet was 4.0, but this fell to 0.30 in ceca and ca. 0.8 in liver, blood, and whole carcass one day after feeding. These results indicate that ceca are active in the synthesis of 22:6n-3 and the oxidation of 18:3n-3.