Pathways of Lipid Metabolism in Marine Algae, Co-Expression Network, Bottlenecks and Candidate Genes for Enhanced Production of EPA and DHA in Species of Chromista (original) (raw)
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Algal Research, 2015
Nannochloropsis oceanica is an important source for omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) such as eicosapentaenoic acid (EPA, 20:5 n − 3), and a potent candidate for biofuel production, due to its outstanding capability for rapid induction of triacylglycerol (TAG) overproduction. In contrast to membrane lipids, TAG of N. oceanica is poor in the valuable LC-PUFA, EPA. We identified, cloned and characterized a N. oceanica microsomal-like Δ12-desaturase (NoD12) mediating the committing step of LC-PUFA biosynthesis by converting oleic acid (18:1 n − 9) to linoleic acid (LA,18:2 n − 6). We generated strains of N. oceanica overexpressing NoD12 under the control of the stress-inducible endogenous lipid droplet surface protein (LDSP) promoter, resulting in robust expression under nitrogen starvation conditions. The overexpression of NoD12 significantly altered fatty acid composition of total lipids and of individual lipid classes, such as a drastic increase in 18:2 proportion in phosphatidylcholine and in TAG was observed under nitrogen starvation. Some LA was converted further toward LC-PUFA resulting in a substantial increase in arachidonic acid (20:4 n − 6) in TAG. Our data demonstrate the feasibility of metabolic engineering to increase LC-PUFA content in the biotechnologically important microalga using native genes and promoters, and provide novel insights into the regulation of LC-PUFA flux to TAG under nitrogen starvation.
Enhanced production of polyunsaturated fatty acid docosahexaenoic acid by thraustochytrid protists
2004
In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 × ACPs. Enzymes possessing one inactive ACP at any position produced ~30% PUFAs compared with the parental enzyme but unexpectedly had ~250% productivity compared with subunit A with 4 × ACPs. Enzymes constructed by replacing the 3 rd ACP with an inactive ACP from another subunit A or ACP-unrelated sequences produced ~100% and ~3% PUFAs compared with the parental 3 rd ACP-inactive enzyme, respectively. These results suggest that both the structure and number of ACP domains are important for PUFA productivity. Polyunsaturated fatty acids (PUFAs, Fig. 1a) including docosahexaenoic acid (DHA; C22:6 ω 3), eicosapentaenoic acid (EPA; C20:5 ω 3), and arachidonic acid (ARA; C20:4 ω 6) are essential components of membrane lipids and for human nutrition. They also have crucial biological activities such as prevention of arteriosclerosis and hyperlipidaemia 1-4. Therefore, the demand for PUFAs is increasing as medical pharmaceuticals and nutritional supplements. Fishes and fish oils have traditionally been the sole PUFA sources. However, there is concern over the availability of PUFAs because of the unstable supply from marine resources and increasing demand. Therefore, alternative and sustainable sources of PUFAs are required. To this end, fermentative processes have been developed using microorganisms such as microalgae, fungi, and engineered yeasts for the production of DHA, ARA, and EPA, respectively 5-7. PUFAs are biosynthesized by two pathways, the aerobic desaturase/elongase pathway and the anaerobic PUFA synthase pathway. In the former pathway, which operates in plants, fungi, microalgae, and bacteria, specific desaturases and elongases catalyse individual desaturation and elongation steps to synthesize PUFAs from oleic acid (C18:1 ω 9) 7. In the latter pathway, which occurs in eukaryotic microalgae and prokaryotic bacteria, PUFA synthases composed of huge enzyme complexes with multiple catalytic domains synthesize PUFAs using acetyl coenzyme A (CoA) and malonyl-CoA as starter and extender units, respectively, in a manner similar to polyketide synthases (PKSs) and fatty acid synthases (FASs) 8-11. From the viewpoint of industrial production of PUFAs, the latter process has great advantages because it requires fewer reducing equivalents such as NADPH and produces smaller amounts of by-products with undesirable chain lengths and unsaturated positions. PUFA synthase genes have been identified as clusters not only in marine microorganisms such as Shewanella oneidensis 12 , Photobacterium profundum 13 , Moritella marina 14 , Aureispira marina 15 , and Schizochytrium sp. 16 , but also in terrestrial myxobacteria 17. All the PUFA synthases identified are huge multifunctional enzyme complexes consisting of three to four subunits. They possess acyltransferase (AT), malonyl-CoA transferase (MAT), ketoacyl
Journal of Lipid Research, 2012
, and their metabolites have attracted increasing attention for the development of medicines and nutritional supplements based on their serological, cardiovascular, and antiinfl ammatory benefi ts (1-3). For example, EPA is used to treat hyperlipidemia and arteriosclerotic diseases. Furthermore, DHA plays important roles in the development of the retina and the brain in infants and possibly in the maintenance of normal brain function in adults (4, 5). Fish oils, such as sardine and tuna oils, are the major commercial sources of EPA and DHA. However, there is a concern that fi sh oils will be insuffi cient to meet the increasing global demand for these PUFAs in the future. Thus, several microorganisms and plants have been explored as alternative sources of PUFAs (6). Thraustochytrids are eukaryotic marine protists, including the typical genera Thraustochytrium and Aurantiochytrium (formerly Schizochytrium), which belong to the Stramenopiles, class Labyrinthulomycetes , family Thraustochytriaceae. These organisms are commonly found in marine and estuarine environments and play important roles in the degradation and mineralization of organic materials in marine ecosystems. Thraustochytrids have recently received increasing attention for their ability to produce and accumulate high amounts of n-3 PUFAs in cellular lipid Abstract Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative ⌬ 12-fatty acid desaturase (Tau ⌬ 12des) from T. aureum. Yeasts transformed with the tau ⌬ 12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tau ⌬ 12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tau ⌬ 12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tau ⌬ 12des-disruption mutants with a tau ⌬ 12des expression cassette restored the wild-type fatty acid profi les. These data clearly indicate that Tau ⌬ 12des functions as ⌬ 12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways .-Matsuda, T.
Proceedings of the Nutrition …, 2005
There is now considerable evidence of the importance of n-3 long-chain PUFA in human health and development. At the same time, the marine fish stocks that serve as the primary sources of these fatty acids are threatened by continued over-exploitation. Thus, there is an urgent need to provide a sustainable alternative source of the n-3 long-chain PUFA normally found in fish oils. The possibility of using transgenic plants genetically engineered to synthesise these important fatty acids has recently been demonstrated. The approaches taken to realise this outcome will be discussed, as will their prospects for providing a sustainable resource for the future.
PUFA biosynthesis pathway in marine scallop Chlamys nobilis Reeve
Journal of agricultural and food chemistry, 2014
Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in important physiological processes. However, the endogenous PUFA biosynthesis pathway is poorly understood in marine bivalves. Previously, a fatty acyl desaturase (Fad) with Δ5 activity was functionally characterized and an elongase termed Elovl2/5 was reported to efficiently elongate 18:2n-6 and 18:3n-3 to 20:2n-6 and 20:3n-3 respectively in Chlamys nobilis. In this study, another elongase and another Fad were identified. Functional characterization in recombinant yeast showed that the newly cloned elongase can elongate 20:4n-6 and 20:5n-3 to C22 and C24, while the newly cloned scallop Fad exhibited a Δ8-desaturation activity, and could desaturate exogenously added PUFA 20:3n-3 and 20:2n-6 to 20:4n-3 and 20:3n-6 respectively, providing the first compelling evidence that noble scallop could de novo biosynthesize 20:5n-3 and 20:4n-6 from PUFA precursors though the "Δ8 pathway". No Δ6 or Δ4 activity was detect...
Modifying the lipid content and composition of plant seeds: engineering the production of LC-PUFA
Applied microbiology and biotechnology, 2015
Omega-3 fatty acids are characterized by a double bond at the third carbon atom from the end of the carbon chain. Latterly, long chain polyunsaturated omega-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexanoic acid (DHA; 22:6 Δ4,7,10,13,16,19), which typically only enter the human diet via the consumption of oily fish, have attracted much attention. The health benefits of the omega-3 LC-PUFAs EPA and DHA are now well established. Given the desire for a sustainable supply of omega-LC-PUFA, efforts have focused on enhancing the composition of vegetable oils to include these important fatty acids. Specifically, EPA and DHA have been the focus of much study, with the ultimate goal of producing a terrestrial plant-based source of these so-called fish oils. Over the last decade, many genes encoding the primary LC-PUFA biosynthetic activities have been identified and characterized. This has allowed the reconstitution of the LC-PUFA biosynthetic pathway in...
The versatility of algae and their lipid metabolism
Biochimie, 2009
Eukaryotic algae are a very diverse group of organisms that are key components of ecosystems ranging from deserts to the Antarctic. They account for over half of the primary production at the base of food chains. The lipids of different classes are varied and contain unusual compounds not found in other phyla. In this short review, we introduce the major cellular lipids and their fatty acids and then describe how the latter (particularly the polyunsaturated fatty acids, PUFAs) are synthesised. The discovery of different elongases and desaturases important for PUFA production is detailed and their application for biotechnology described. Finally, the potential for algae in commercial applications is discussed, particularly in relation to the production of very long chain PUFAs and biofuel.
Journal of the American Oil Chemists' Society, 2012
Long chain (C C 20) polyunsaturated fatty acids (LC-PUFAs) represent important components of the human diet. Currently, the predominant sources of these fatty acids are marine fish and algal oils, but high production costs and diminishing feedstock, limit their supply and usage. A more regular sustainable source of these compounds is urgently required and therefore research is being conducted to develop a sustainable, land-based production system. This work describes the metabolic engineering of an artificial pathway that activates the production of C 22-PUFAs, docosatetraenoic acid or adrenic acid (ADA) and n-3 docosapentaenoic acid (DPA) in Physcomitrella patens using a gene from a marine algae Pavlova sp. encoding D 5-elongase and vegetable oil supplementation. The accumulation of ADA and x-3 DPA were dramatically increased to 24.3 and 11.7 mg L-1 and accounted for 2.3 and 1.1% of total fatty acids, respectively. This is the first report on producing n-3 DPA, DHA precursor, in P. patens. The obtained results prove that this enzyme appears to be more active when fused to a green fluorescence protein reporter gene. These finding reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation and nutritional supplementation, to produce specific PUFAs in a non-seed lower plant, is a promising technique.
Metabolic Engineering, 2013
An iterative approach to optimising the accumulation of non-native long chain polyunsaturated fatty acids in transgenic plants was undertaken in Arabidopsis thaliana. The contribution of a number of different transgene enzyme activities was systematically determined, as was the contribution of endogenous fatty acid metabolism. Successive iterations were informed by lipidomic analysis of neutral, polar and acyl-CoA pools. This approach allowed for a four-fold improvement on levels previously reported for the accumulation of eicosapentaenoic acid in Arabidopsis seeds and also facilitated the successful engineering of the high value polyunsaturated fatty acid docosahexaenoic acid to 10-fold higher levels. Our studies identify the minimal gene set required to direct the efficient synthesis of these fatty acids in transgenic seed oil.