转基因植物生产超长链多不饱和脂肪酸研究进展

超长链多不饱和脂肪酸(VLCPUFAs)对人类健康非常重要。日常摄入一定量的VLCPUFAs能够补充人体自身合成的不足, 并对某些疾病起到明显的预防和治疗作用。VLCPUFAs主要源自深海鱼油, 但由于市场需求的迅速增长和海洋可捕捞 鱼类资源的日益减少, 该途径已经远远不能满足市场的需要, 寻找更为持续且稳定的VLCPUFAs来源已经成为当务之急。最近, 人们已经克隆了VLCPUFAs生物合成相关的去饱和酶和延伸酶基因, 并希望在植物特别是油料作物中共表达这些基因, 使其成为生产VLCPUFAs的“绿色细胞工厂”。目前已有多个研究小组在进行转基因植物合成VLCPUFAs的探索, 并取得了突破性的研究成果。本文综述了相关的研究进展, 并对存在的问题和解决策略进行了探讨。     

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.     

Metabolic Engineering of Plants to Produce Very Long-chain Polyunsaturated Fatty Acids

Very long-chain polyunsaturated fatty acids (VLCPUFAs) are essential for human health and well-being. However, the current sources of these valuable compounds are limited and may not be sustainable in the long term. Recently, considerable progress has been made in identifying genes involved in the biosynthesis of VLCPUFAs. The co-expression of these genes in model systems such as plant embryos or yeast provided many valuable insights into the mechanisms of VLCPUFA synthesis. The recent successful reconstitution of pathways leading to the synthesis of arachidonic acid, eicosapentaenoic acid and finally docosahexaenoic acid in oil-seed plants indicates the feasibility of using transgenic crops as alternative sources of VLCPUFAs. The various approaches used to attain these results and the specific constraints associated with each approach are discussed.     

Molecular mechanisms for biosynthesis and assembly of nutritionally important very long chain polyunsaturated fatty acids in microorganisms

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3), arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5-n3) are nutritionally important for humans and animals. De novo biosynthesis of these fatty acids mainly occurs in microorganisms and goes through either an aerobic pathway catalyzed by type I/II fatty acid synthase, desaturases and elongases or an anaerobic pathway catalyzed by a polyunsaturated fatty acid synthase. After synthesis, VLCPUFAs must be incorporated into glycerolipids for storage through acyl assembly processes. Understanding the mechanisms for the biosynthesis of VLCPUFAs and their incorporation into glycerolipids is important not only for developing a renewable, sustainable and environment-friendly source of these fatty acids in microorganisms, but also, for designing effective strategies for metabolic engineering of these fatty acids in heterologous systems. This review highlights recent findings which have increased our understanding of biosynthesis of VLCPUFAs and their incorporation into glycerolipids in microorganisms. Future directions in improving the production of VLCPUFAs in native microbial producers are also discussed along with transgenic production of these fatty acids in oleaginous microorganisms and oilseed crops for food and feed uses.     

Metabolic engineering of omega3-very long chain polyunsaturated fatty acid production by an exclusively acyl-CoA-dependent pathway

omega3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Delta6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds.     

Production of eicosapentaenoic acid (EPA, 20:5n-3) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) through the alternative ∆8 desaturation pathway mediated by particle bombardment

Very long chain polyunsaturated fatty acids (VLCPUFAs), especially eicosapentaenoic acid (EPA, 20:5?^5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6?^{4,7,10,13,16,19}) have demonstrated important roles in a number of aspects of human health. Currently, our primary dietary sources for these fatty acids are from marine fish. Producing VLCPUFAs in oilseed crop by metabolic engineering was considered to provide an alternative, sustainable sources. Here, three heterologous genes, ?9 elongase (?9-Elo) of Isochrysis galbana, ?8 desaturase (?8-Des) of Euglena gracilis, ?5 desaturase (?5-Des) of Mortierella alpina, were co-transformed into maize inbred line Qi319 using a particle bombardment transformation method to produce EPA through the alternative ?8 desaturation synthetic pathway. A total of 144 herbicide resistant lines were obtained with an average transformation efficiency of 7.95%, of which 98 lines contain the Bar genes with the positive transformation efficiency of 4.74%. In addition, 60 of the 98 positive transgenic lines were identified to contain all three transgenes, ?9-Elo, ?8-Des, and ?5-Des. The fatty acid composition of the leaves from the 60 transgenic lines were subjected to gas liquid chromatography analysis and the results showed that the amounts of EPA reached 1.99% in an individual leaf. These data demonstrate the feasibility for the heterologous production of EPA in maize and this will lay a foundation for the production of VLCPUFAs, including EPA and DHA, in maize by metabolic engineering in the future.     

球等鞭金藻中Δ5去饱和酶基因的克隆与功能鉴定

球等鞭金藻(Isochrysis galbana)是一类单细胞海洋微藻,富含二十二碳六烯酸(DHA,22:6Δ4,7,10,13,16,19)。我们利用RACE的方法从球等鞭金藻cDNA文库中同源克隆到一个大小为1329 bp的cDNA片段,编码442个氨基酸的多肽,分子量约49.9 kD。生物信息学分析表明,其编码产物N端具有细胞色素b5结构域,以及与电子传递有关的三个富含组氨酸的结构域,与Pavlova salinaΔ5去饱和酶同源性最高,达56%,故将该基因命名为IgD5。酿酒酵母功能鉴定实验表明,其编码的蛋白质具有Δ5去饱和酶活性,能够将二高-γ-亚麻酸(DGLA,20:3Δ8,11,14)转化成花生四烯酸(AA,20:4Δ5,8,11,14),转化效率平均为34.6%,最高可达40.3%。     

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits. We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important omega-3 fatty acid in oilseed crops.     

Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: solving bottlenecks in fatty acid flux

Oilseeds provide a unique platform for the production of high-value fatty acids that can replace non-sustainable petroleum and oceanic sources of specialty chemicals and aquaculture feed. However, recent efforts to engineer the seeds of crop and model plant species to produce new types of fatty acids, including hydroxy and conjugated fatty acids for industrial uses and long-chain omega-3 polyunsaturated fatty acids for farmed fish feed, have met with only modest success. The collective results from these studies point to metabolic 'bottlenecks' in the engineered plant seeds that substantially limit the efficient or selective flux of unusual fatty acids between different substrate pools and ultimately into storage triacylglycerol. Evidence is emerging that diacylglycerol acyltransferase 2, which catalyzes the final step in triacylglycerol assembly, is an important contributor to the synthesis of unusual fatty acid-containing oils, and is likely to be a key target for future oilseed metabolic engineering efforts.     

High-value oils from plants

The seed oils of domesticated oilseed crops are major agricultural commodities that are used primarily for nutritional applications, but in recent years there has been increasing use of these oils for production of biofuels and chemical feedstocks. This is being driven in part by the rapidly rising costs of petroleum, increased concern about the environmental impact of using fossil oil, and the need to develop renewable domestic sources of fuel and industrial raw materials. There is also a need to develop sustainable sources of nutritionally important fatty acids such as those that are typically derived from fish oil. Plant oils can provide renewable sources of high-value fatty acids for both the chemical and health-related industries. The value and application of an oil are determined largely by its fatty acid composition, and while most vegetable oils contain just five basic fatty acid structures, there is a rich diversity of fatty acids present in nature, many of which have potential usage in industry. In this review, we describe several areas where plant oils can have a significant impact on the emerging bioeconomy and the types of fatty acids that are required in these various applications. We also outline the current understanding of the underlying biochemical and molecular mechanisms of seed oil production, and the challenges and potential in translating this knowledge into the rational design and engineering of crop plants to produce high-value oils in plant seeds.     

Plants as 'chemical factories' for the production of polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are valuable products because of their involvement in several aspects of human health. Market demand for most PUFAs is growing continually and current sources are considered insufficient for satisfying this demand; alternative sources are actively sought after. Oilseed plants can be a potential source of PUFAs if they are appropriately gene engineered. Most of the basic tools for genetic engineering of oilseed plants for giving them the ability to produce PUFAs are already developed. Here we review the prospects of genetic engineering of oilseed plants for producing some valuable long-chain polyunsaturated fatty acids. Genetic transformation for GLA production seems to be a near-term possibility, but gene engineering seems considerably more difficult for the other long-chain PUFAs. Nevertheless, with the current rapid pace of biotechnological advancement, the remaining difficulties may be surmounted in the near future.     

Metabolic engineering of fatty acid biosynthesis in plants.

Fatty acids are the most abundant form of reduced carbon chains available from nature and have diverse uses ranging from food to industrial feedstocks. Plants represent a significant renewable source of fatty acids because many species accumulate them in the form of triacylglycerol as major storage components in seeds. With the advent of plant transformation technology, metabolic engineering of oilseed fatty acids has become possible and transgenic plant oils represent some of the first successes in design of modified plant products. Directed gene down-regulation strategies have enabled the specific tailoring of common fatty acids in several oilseed crops. In addition, transfer of novel fatty acid biosynthetic genes from noncommercial plants has allowed the production of novel oil compositions in oilseed crops. These and future endeavors aim to produce seeds higher in oil content as well as new oils that are more stable, are healthier for humans, and can serve as a renewable source of industrial commodities. Large-scale new industrial uses of engineered plant oils are on the horizon but will require a better understanding of factors that limit the accumulation of unusual fatty acid structures in seeds.     

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.     

Metabolic engineering of edible plant oils]

Plant seed oil is the major source of many fatty acids for human nutrition, and also one of industrial feedstocks. Recent advances in understanding of the basic biochemistry of seed oil biosynthesis, coupled with cloning of the genes encoding the enzymes involved in fatty acid modification and oil accumulation, have set the stage for the metabolic engineering of oilseed crops that produce "designer" plant seed oils with the improved nutritional values for human being. In this review we provide an overview of seed oil biosynthesis/regulation and highlight the key enzymatic steps that are targets for gene manipulation. The strategies of metabolic engineering of fatty acids in oilseeds, including overexpression or suppression of genes encoding single or multi-step biosynthetic pathways and assembling the complete pathway for the synthesis of long-chain polyunsaturated fatty acids (e.g. arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid) are described in detail. The current "bottlenecks" in using common oilseeds as "bioreactors" for commercial production of high-value fatty acids are analyzed. It is also discussed that the future research focuses of oilseed metabolic engineering and the prospects in creating renewable sources and promoting the sustainable development of human society and economy.     

Rational metabolic engineering of transgenic plants for biosynthesis of omega-3 polyunsaturates

The health-beneficial effects of long-chain polyunsaturated fatty acids (LC-PUFAs), derived mainly from fish oil, coupled with the growing requirement for an alternative and sustainable source of these compounds, has led to efforts to engineer oilseed crops for their production. LC-PUFA synthesis has been achieved using combinations of heterologous endomembrane desaturases and elongases expressed in model oilseed plants. Two general approaches have been employed that both use endogenous 18 carbon fatty acids as the starting substrates: the Delta6- and Delta8-pathways, which perform desaturation followed by elongation or elongation followed by desaturation, respectively. However, yields above 20% have not yet been realized owing to bottlenecks that become apparent in the endogenous biosynthetic pathways when heterologous genes are expressed. These bottlenecks might be caused partly by inefficient non-native enzymes in the host system or also by suboptimal acyl-exchange mechanisms between the acyl-CoA and lipid class pools. The fine-tuning of the fatty acid flux between the acyl-CoA, phospholipid, and triacylglycerol pools will be essential to maximise polyunsaturated fatty acid yields in seed oils. In addition, efficient substrate channelling and lipid synthesis could depend on specific endoplasmic reticulum subdomain localisation for key endogenous enzymes, and this organization could be compromised in heterologous systems.     

Polyunsaturated fatty acids: biotechnology

Polyunsaturated fatty acids like EPA and DHA have attracted a great attention due to their beneficial effects on human health. At present, fish oil is the major source of EPA and DHA. Various alternative sources are being explored to get these essential fatty acids. Genes encoding enzymes involved in the biosyntheses of PUFAs have been identified, cloned and gene prospecting becomes a novel method for enhanced PUFA production. Desaturase and elongase genes have important biotechnological appeal from genetic engineering point of view. This review highlights the research and results on such enzymes.     

Polyunsaturated Fatty Acids: Biotechnology

ABSTRACT

Polyunsaturated fatty acids like EPA and DHA have attracted a great attention due to their beneficial effects on human health. At present, fish oil is the major source of EPA and DHA. Various alternative sources are being explored to get these essential fatty acids. Genes encoding enzymes involved in the biosyntheses of PUFAs have been identified, cloned and gene prospecting becomes a novel method for enhanced PUFA production. Desaturase and elongase genes have important biotechnological appeal from genetic engineering point of view. This review highlights the research and results on such enzymes.     

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.     

Engineering oilseeds to produce nutritional fatty acids

There is a growing body of evidence suggesting that regular consumption of foods rich in omega-3 long chain polyunsaturated fatty acids has multiple positive health benefits. The fats and oils from marine fish contain high contents of these beneficial fatty acids but increased consumer demand has also increased strain on the ability of the world's fisheries to meet demand from wild capture. Many consumers are choosing fish oil supplements or are eating foods that have been complemented with fish oils instead of consuming fish directly. However, removing undesirable odors, flavors and contaminants is expensive. In contrast, oils derived from land plants such as soybean are inexpensive and contaminant free. Recent strides in plant molecular biology now allow the engineering of oilseeds for the production of novel fats and oils, including those synthesized by complex, multigene biosynthetic pathways such as the omega-3 long chain polyunsaturated fatty acids. Given the potential benefits to the environment with regards to overfishing and the health prospects of increased consumption of these healthy fatty acids, producing these fatty acids in oilseeds is a desirable and worthy goal. In this review, we will describe the recent advances in this field along with some of the technical hurdles encountered thus far.