Biotechnology and the improvement of oil crops – genes,dreams and realities

During the past decade, there have been many optimistic claims concerning the potential of novel oil-based products from genetically engineered crops, particularly for the manufacture of a new generation of renewable, carbon-neutral, industrial materials. Such claims have been underpinned by an impressive series of scientific advances that have resulted in the isolation of genes encoding most of the enzymes directly involved in oil biosynthesis. In some cases, these enzymes have even been re-engineered by site-directed or random mutagenesis to allow production of new fatty acid profiles that are not present in any existing organism. This has opened up the prospect of engineering `designer oil crops' to produce novel fatty acids with chain lengths from C8 to C24 and with a wide range of industrially useful functionalities, including hydroxylation, epoxidation, and conjugated and non-conjugated double or triple bonds. However, there remain significant technical challenges before this promise of designer transgenic crops is likely to be translated into large-scale commercial reality. For example, it has proved surprisingly difficult to engineer high levels of novel fatty acids in genetically engineered transgenic plants, although many wild type seeds can readily accumulate 90–95% of a single fatty acid in their storage oil. Another complication is the recent discovery of multiple pathways of triacylglycerol biosynthesis and the difficulty in ensuring that novel fatty acids are only channelled towards storage triacylglycerols and not to membrane or signalling lipids in major target crops like rapeseed. New findings from our lab have suggested that there may also be problems with the tissue specificity of some of the `seed-specific' gene promoters that are commonly used in transgenic crops. There are also considerable and often underestimated challenges associated with the economics, management and public acceptability of all transgenic crops, even for non-food use. In most cases the projections of petroleum reserves over the next few decades make it unlikely that crop-derived commodity products that substitute for petroleum will be competitive. Also the scale of crop production required to generate millions of tonnes of commodity oils, e.g., for biodegradable plastics, is likely to seriously impinge on food production at a time of increasing global populations, and is therefore unlikely to be acceptable. An alternative strategy to transgenic oil crops is to use molecular breeding techniques in order to develop new crops that already synthesise high levels of novel fatty acids of interest. Finally, the most promising market sectors and product ranges for the future development of oil crop biotechnology will be discussed.     

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.     

Engineering plant oils as high-value industrial feedstocks for biorefining: the need for underpinning cell biology research

Plant oils represent renewable sources of long-chain hydrocarbons that can be used as both fuel and chemical feedstocks, and genetic engineering offers an opportunity to create further high-value specialty oils for specific industrial uses. While many genes have been identified for the production of industrially important fatty acids, expression of these genes in transgenic plants has routinely resulted in a low accumulation of the desired fatty acids, indicating that significantly more knowledge of seed oil production is required before any future rational engineering designs are attempted. Here, we provide an overview of the cellular features of fatty acid desaturases, the so-called diverged desaturases, and diacylglycerol acyltransferases, three sets of enzymes that play a central role in determining the types and amounts of fatty acids that are present in seed oil, and as such, the final application and value of the oil. Recent studies of the intracellular trafficking, assembly and regulation of these enzymes have provided new insights to the mechanisms of storage oil production, and suggest that the compartmentalization of enzyme activities within specific regions or subdomains of the ER may be essential for both the synthesis of novel fatty acid structures and the channeling of these important fatty acids into seed storage oils.     

新型可再生工业用油脂的代谢工程

植物种子油是一种可再生资源,亦用作生物燃油和化学工业原料. 一些野生植物能高水平合成积累羟化、环氧化和共轭脂肪酸等具有重要工业应用价值的特异脂肪酸.催化这些特异脂肪酸合成的酶主要是类脂肪酸去胞和酶2(类FAD2). 由特异脂肪酸合成到三酰基甘油脂 (TAG) 形成还需要酰基转移酶 (如DGAT) 的参与. 在油料作物种子中表达类FAD2酶及其相关基因(如DGAT),已培育出了能合成积累一定含量特异脂肪酸的工程油料品系,为基于农作物生产高附加值工业用油脂开辟了新途径. 本文论述了参与特异脂肪酸生物合成途径的关键酶基因、油料作物代谢工程策略,以及应用工程油料作物大规模生产重要工业用脂肪酸的研究进展、存在问题和应用前景等.     

FAD2类酶在合成工业用脂肪酸中的应用

许多工业用稀有脂肪酸存在于非食用植物种子油中,它们由不同脂肪酸Δ12-去饱和酶（FAD2）催化,在油酸Δ12位引入环氧基、羟基、形成三键或共扼双键。目前已从不同生物中克隆得到一系列FAD2酶基因,并在油料植物中获得成功表达。但总体上看,目标脂肪酸累积量还相对较低,稀有脂肪酸生物合成及其从磷脂酰胆碱（PC）到储存甘油三酯(IAG)的转化机制尚需要进一步阐明。     

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.     

Metabolic engineering of plants for polyunsaturated fatty acid production

Very-long-chain polyunsaturated fatty acids (VLCPUFAs) have demonstrated health benefits. Currently, the main sources for these fatty acids are oils from fish and microbes. However, shrinking fish populations and the high cost of microbial oil extraction are making the economic sustainability of these sources questionable. Metabolic engineering of oilseed crops could provide a novel and sustainable source of VLCPUFAs. Recently, genes encoding desaturases and elongases from microbes have been identified and successfully expressed in oilseed plants. However, the levels of VLCPUFAs produced in transgenic plants expressing these genes are still much lower than those found in native microbes. This review assesses the recent progress and future perspectives in the metabolic engineering of PUFAs in plants.     

Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil

SUMMARY: A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from many wild species, progress has been limited because the expression of these genes in transgenic plants produces low yields of the desired products. For example, Ricinus communis fatty acid hydroxylase 12 (FAH12) produces a maximum of only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis. cDNA clones encoding R. communis enzymes for additional steps in the seed oil biosynthetic pathway were identified. Expression of these cDNAs in FAH12 transgenic plants revealed that the R. communis type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2) could increase HFAs from 17% to nearly 30%. Detailed comparisons of seed neutral lipids from the single- and double-transgenic lines indicated that RcDGAT2 substantially modified the triacylglycerol (TAG) pool, with significant increases in most of the major TAG species observed in native castor bean oil. These data suggest that RcDGAT2 prefers acyl-coenzyme A and diacylglycerol substrates containing HFAs, and biochemical analyses of RcDGAT2 expressed in yeast cells confirmed a strong preference for HFA-containing diacylglycerol substrates. Our results demonstrate that pathway engineering approaches can be used successfully to increase the yields of industrial feedstocks in plants, and that members of the DGAT2 gene family probably play a key role in this process.     

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.     

Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation

Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products.     

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.     

An alternative to fish oils: Metabolic engineering of oil-seed crops to produce omega-3 long chain polyunsaturated fatty acids

It is now accepted that omega-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) play important roles in a number of aspects of human health, with marine fish rich in these beneficial fatty acids our primary dietary source. However, over-fishing and concerns about pollution of the marine environment indicate a need to develop alternative, sustainable sources of very long chain polyunsaturated fatty acids (VLC-PUFAs) such as EPA and DHA. A number of different strategies have been considered, with one of the most promising being transgenic plants “reverse-engineered” to produce these so-called fish oils. Considerable progress has been made towards this goal and in this review we will outline the recent achievements in demonstrating the production of omega-3 VLC-PUFAs in transgenic plants. We will also consider how these enriched oils will allow the development of nutritionally-enhanced food products, suitable either for direct human ingestion or for use as an animal feedstuff. In particular, the requirements of aquaculture for omega-3 VLC-PUFAs will act as a strong driver for the development of such products. In addition, biotechnological research on the synthesis of VLC-PUFAs has provided new insights into the complexities of acyl-channelling and triacylglycerol biosynthesis in higher plants.     

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.     

Identification of bottlenecks in the accumulation of cyclic fatty acids in camelina seed oil

Modified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA‐producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural‐occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co‐expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA‐accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co‐expression of SfLPAT with EcCPS, di‐CPA‐PC increased by ~50% relative to lines expressing EcCPS alone with the di‐CPA‐PC primarily observed in the embryonic axis and mono‐CPA‐PC primarily in cotyledon tissue. EcCPS‐SfLPAT lines revealed a redistribution of CPA from the sn‐1 to sn‐2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.     

Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis

Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. However, expression of the enzymes that catalyze the primary synthesis of these unusual FAs in transgenic plants typically results in low levels of the desired FA. For example, seed-specific expression of castor (Ricinus communis) fatty acid hydroxylase (RcFAH) in Arabidopsis (Arabidopsis thaliana) resulted in only 17% hydroxy fatty acids (HFAs) in the seed oil. In order to increase HFA levels, we investigated castor phospholipid:diacylglycerol acyltransferase (PDAT). We cloned cDNAs encoding three putative PDAT enzymes from a castor seed cDNA library and coexpressed them with RcFAH12. One isoform, RcPDAT1A, increased HFA levels to 27%. Analysis of HFA-triacylglycerol molecular species and regiochemistry, along with analysis of the HFA content of phosphatidylcholine, indicates that RcPDAT1A functions as a PDAT in vivo. Expression of RcFAH12 alone leads to a significant decrease in FA content of seeds. Coexpression of RcPDAT1A and RcDGAT2 (for diacylglycerol acyltransferase 2) with RcFAH12 restored FA levels to nearly wild-type levels, and this was accompanied by a major increase in the mass of HFAs accumulating in the seeds. We show the usefulness of RcPDAT1A for engineering plants with high levels of HFAs and alleviating bottlenecks due to the production of unusual FAs in transgenic oilseeds.     

基因工程在改善植物油营养价值中的应用

本文介绍了近年来应用基因工程技术对植物油的脂肪酸成分进行改造,进而生产有益于健康的食用油所取得的进展.近10年来,应用基因修饰的方法对植物油进行营养学方面的改良已经取得了很大的进展,随着植物脂肪酸生物合成途径的日趋明确,通过转基因技术可以让植物生产许多含有特殊脂肪酸成分且对人类健康有益的植物油.     

基因工程在改善植物油营养价值中的应用

本文介绍了近年来应用基因工程技术对植物油的脂肪酸成分进行改造, 进而生产有益于健康的食用油所取得的进展。近10年来, 应用基因修饰的方法对植物油进行营养学方面的改良已经取得了很大的进展, 随着植物脂肪酸生物合成途径的日趋明确, 通过转基因技术可以让植物生产许多含有特殊脂肪酸成分且对人类健康有益的植物油。     

Metabolic engineering of new fatty acids in plants

Metabolic engineering of plants to express high levels of new fatty acids that are of nutritional and industrial importance has proven to be highly challenging. Significant advances have been made recently, however, particularly in the development of the first plant oils to contain long-chain polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. Methods of increasing the accumulation of Delta12-modified fatty acids synthesized by transgenically expressed FAD2-like enzymes have also been investigated. Biochemical analyses of plants that express these introduced fatty-acid metabolic pathways have highlighted the central importance of ensuring the removal of novel fatty acids from their site of synthesis on phosphatidylcholine to enable their further modification, exclusion from membrane lipids and accumulation in seed triacylglycerols.     

Industrial oils from transgenic plants

Unusual fatty acids that have useful industrial properties occur widely in the seed oils of many non-agronomic plant species. Researchers are attempting to use biotechnology to produce high levels of these fatty acids in the seeds of existing crop plants. cDNAs for a wide variety of unusual fatty acid biosynthetic enzymes have been identified, particularly through the use of expressed sequence tags. However, it has not yet been possible to use these cDNAs to produce large amounts of unusual fatty acids in seeds of transgenic plants. This difficulty points to the need for a greater understanding of fatty acid metabolism in oilseeds.