食用植物油脂的代谢工程

植物种子油可提供人类营养所需的多种脂肪酸,也是工业用油的原料之一。文章结合我们对植物种子发育、脂肪酸生物合成途径和大豆油脂遗传改良的研究,重点论述参与脂肪酸合成及其调控的一些关键酶的基因、代谢工程改良植物油脂营养价值的技术策略及其研究进展,分析目前应用油料作物种子作为“生物反应器”规模化生产有重要营养价值和特殊用途的脂肪酸的问题及技术“瓶颈”,讨论未来植物脂肪酸代谢工程主攻方向以及在培育可再生资源和推动人类社会及经济可持续发展中的应用前景。     

沙棘种子高积累碳十八不饱和脂肪酸的多基因协同调控机制

为探讨沙棘种子油高积累碳十八不饱和脂肪酸的多基因协同作用机制,以近缘低油沙棘品系‘绥棘1号’和高油品系‘新俄3号’6个不同发育期的种子为材料,利用气相色谱飞行时间质谱法测定种子油脂肪酸组份,采用qRT-PCR方法分析不饱和脂肪酸合成积累相关基因KAR、FATB、Δ9 D、KASⅡ、SAD、FAD2、FAD3、FAD7和FAD8的表达模式,验证多基因表达对碳十八不饱和脂肪酸积累的影响。结果表明:(1)‘绥棘1号’和‘新俄3号’种子油均高积累碳十八不饱和脂肪酸,分别占总脂肪酸的87.71%和88.68%;种子发育期间,油酸相对含量一直呈上升趋势,亚油酸相对含量短时下降后上升趋稳,而亚麻酸相对含量则呈先上升后下降趋稳。(2)FATB基因下调表达协同Δ9 D基因低表达,使C16∶0-ACP转化为棕榈酸和棕榈油酸的代谢减弱,而KAR和KASⅡ基因的相对上调表达,促进了硬脂酸合成,为碳十八不饱和脂肪酸的合成积累了较多前体。(3)SAD基因的持续高表达催化硬脂酸去饱和为油酸,且持续上升的SAD/FATB基因表达比直接提高了脂肪酸的去饱和速率;FAD2、FAD3、FAD7和FAD8基因在亚油酸和亚麻酸快速合成期间同时出现明显的表达量峰值,进而促进油酸逐步去饱和为亚油酸和亚麻酸。研究认为,沙棘种子油高积累碳十八不饱和脂肪酸源于FATB和Δ9 D基因的低表达及KAR、KASⅡ、SAD、FAD2、FAD3、FAD7和FAD8基因的协同高表达,本研究结果为进一步理解种子油中碳十八不饱和脂肪酸的合成积累提供了理论依据,对改良植物油脂的不同脂肪酸比具有重要意义。     

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.     

植物ω-7脂肪酸的系统代谢工程

ω-7脂肪酸（C16：1△9,C18：1△11,C20：1△13）,特别是棕榈油酸（C16：1△9）具有重要的工业、营养和医药价值。这些珍稀脂肪酸大多在一些野生植物的种子中合成,不能商业化生产。对普通油料作物的油脂代谢途径进行遗传修饰,使其种子大量合成并积累ω-7脂肪酸,已成为生物技术和可再生资源研究的一个热点领域。基因操作的主要靶标包括：不同来源的△9脱氢酶的应用、提高底物（C16：0）的浓度、共表达质体型和内质网型？9脱氢酶以及代谢物流的优化等。该文在解析ω-7脂肪酸生物合成途径及其调控网络的基础上,重点论述了ω-7脂肪酸代谢工程的技术策略、研究进展和存在的问题,并进一步讨论了油脂物组学和转基因组学等组学技术在鉴定参与ω-7脂肪酸生物合成途径及其调控的特异基因和优化油脂代谢工程设计上的应用前景。     

地菍果实成熟过程中品质及种子油脂肪酸组分的变化

对地菍果实不同成熟期品质的研究表明,成熟时的地菍果实,维生素C、总糖含量达最高值,酸度则降至最低。此期风味最好,软硬适宜,色泽甚佳,为地菍鲜食的最佳采收期。对不同成熟期的种子油脂肪酸分析表明,种子油以不饱和脂肪酸为主,尤其是亚油酸含量很高,达84%;果实成熟过程中脂肪酸各级分的含量变化趋势不明显。     

紫苏种子脂肪酸组成及合成代谢研究进展

紫苏是一种新型油料作物,种子含油量为35%左右,紫苏籽油脂肪酸组成丰富,含有棕榈酸(16:0)、硬脂酸(18:0)、油酸(18:1)、亚油酸(18:2)和α-亚麻酸(18:3)等,其中α-亚麻酸(ALA)含量高达60%,广泛用于功能性保健食品、药物及油脂化工业.介绍紫苏种子脂肪酸组成及合成代谢基本途径,对近年来脂肪酸合成代谢基因工程研究进行概述与展望.     

植物ω-7脂肪酸的系统代谢工程

ω-7脂肪酸(C16:1Δ9, C18:1Δ11, C20:1Δ13), 特别是棕榈油酸(C16:1Δ9)具有重要的工业、营养和医药价值。这些珍稀脂肪酸大多在一些野生植物的种子中合成, 不能商业化生产。对普通油料作物的油脂代谢途径进行遗传修饰, 使其种子大量合成并积累ω-7脂肪酸, 已成为生物技术和可再生资源研究的一个热点领域。基因操作的主要靶标包括: 不同来源的Δ9脱氢酶的应用、提高底物(C16:0)的浓度、共表达质体型和内质网型Δ9脱氢酶以及代谢物流的优化等。该文在解析ω-7脂肪酸生物合成途径及其调控网络的基础上, 重点论述了ω-7脂肪酸代谢工程的技术策略、研究进展和存在的问题, 并进一步讨论了油脂物组学和转基因组学等组学技术在鉴定参与ω-7脂肪酸生物合成途径及其调控的特异基因和优化油脂代谢工程设计上的应用前景。     

木兰科四种植物种子油的提取及脂肪酸成分分析

采用超声波辅助提取法和微波辅助提取法同时提取白玉兰、凹叶厚朴、深山含笑和醉香含笑四种木兰科植物的种子油,种子油甲酯化后,运用气相色谱—质谱联用技术测定其脂肪酸成分。结果表明:四种植物种子油的提取率不同,白玉兰平均为27.35%、凹叶厚朴23.34%、深山含笑31.66%,醉香含笑9.27%。不同提取方法所得到的种子油脂肪酸成分和相对含量不同,但四种种子油的主要脂肪酸成分相同,包括油酸、亚油酸、硬脂酸和棕榈酸。     

植物二酰甘油酰基转移酶基因(DGAT)研究进展

三酰甘油(TAG)是油料作物最主要的储藏脂类,二酰甘油酰基转移酶(DGAT,EC2.3.1.20)是TAG合成途径的限速酶,其主要作用是催化二酰甘油加上酰基脂肪酸形成三酰甘油.在植物中已发现了3种不同类型的DGAT基因,分别为DGAT1、DGAT2和DGAT3.该文对近年来国内外有关植物DGAT相关基因及其蛋白分类、定位、结构及其在脂肪酸合成、种子发育与萌发、幼苗发育、叶片新陈代谢等过程中的作用等研究进展进行综述.为提高油料作物种子油含量以及特定脂肪酸积累提供理论参考.     

Calgene开发中链脂肪酸新来源

Calgene研究科学家Aubrey Jones在美国植物生理协会的年度会议上提交了一篇论文报告。文章指出,他们应用来自植物种萼距花克隆的Cuph-2中链硫酯酶基因在canola植物中已得到成功表达。该植物累积的种子油通常占总的种子油(即C8～C10的中链脂肪酸(MCFAs))量的75%。本文是高产C8和C10脂肪酸的关键酶在canola中遗传表达的首次报道。目前,该方法已广泛应用于合成润滑剂,食品,以及医学上的营养和药用产品。     

Intraspecific and interspecific adaptive latitudinal cline in Brassicaceae seed oil traits

Premise of the Study

Nearly all seed plants rely on stored seed reserves before photosynthesis can commence. Natural selection for seed oil traits must have occurred over 319 million years of evolution since the first seed plant ancestor. Accounting for the biogeographic distribution of seed oil traits is fundamental to understanding the mechanisms of adaptive evolution in seed plants. However, the evolution of seed oils is poorly understood. We provide evidence of the adaptive nature of seed oil traits at the intraspecific and interspecific levels in Brassicaceae—an oilseed‐rich and economically important plant family.

Methods

Univariate statistics, Pearson's correlation, multiple regression, generalized linear mixed model analysis, and phylogenetic autocorrelation tests on seed oil traits of 360 accessions of Arabidopsis thaliana and 216 Brassicaceae species helped provide evidence of the adaptive nature of seed oil traits.

Key Results

At higher latitudes, both seed oil content and unsaturated fatty acids have selective advantages in Arabidopsis thaliana (intraspecific‐level), while only unsaturated fatty acids have selective advantages across 216 Brassicaceae species (interspecific‐level). The seed oil patterns fit within the theoretical framework of the gradient model. Seed oil content increases significantly from temperate to subtropical to tropical regions in Brassicaceae herbs. Absence of phylogenetic signals for seed oil traits and high seed oil content in four tribes of Brassicaceae were observed.

Conclusions

Multiple seed oil traits are adaptive in nature and follow a gradient model. Consistent evolutionary patterns of seed oil traits were observed at the intraspecific and interspecific levels in Brassicaceae. Seed oil traits change with latitude and across biomes, suggesting selection. The absence of a phylogenetic signal for seed oil traits and the occurrence of high seed oil content in four Brassicaceae tribes provides evidence of the adaptive nature of seed oil traits in Brassicaceae.     

Genetic enhancement of palmitic acid accumulation in cotton seed oil through RNAi down‐regulation of ghKAS2 encoding β‐ketoacyl‐ACP synthase II (KASII)

Palmitic acid (C16:0) already makes up approximately 25% of the total fatty acids in the conventional cotton seed oil. However, further enhancements in palmitic acid content at the expense of the predominant unsaturated fatty acids would provide increased oxidative stability of cotton seed oil and also impart the high melting point required for making margarine, shortening and confectionary products free of trans fatty acids. Seed‐specific RNAi‐mediated down‐regulation of β‐ketoacyl‐ACP synthase II (KASII) catalysing the elongation of palmitoyl‐ACP to stearoyl‐ACP has succeeded in dramatically increasing the C16 fatty acid content of cotton seed oil to well beyond its natural limits, reaching up to 65% of total fatty acids. The elevated C16 levels were comprised of predominantly palmitic acid (C16:0, 51%) and to a lesser extent palmitoleic acid (C16:1, 11%) and hexadecadienoic acid (C16:2, 3%), and were stably inherited. Despite of the dramatic alteration of fatty acid composition and a slight yet significant reduction in oil content in these high‐palmitic (HP) lines, seed germination remained unaffected. Regiochemical analysis of triacylglycerols (TAG) showed that the increased levels of palmitic acid mainly occurred at the outer positions, while C16:1 and C16:2 were predominantly found in the sn‐2 position in both TAG and phosphatidylcholine. Crossing the HP line with previously created high‐oleic (HO) and high‐stearic (HS) genotypes demonstrated that HP and HO traits could be achieved simultaneously; however, elevation of stearic acid was hindered in the presence of high level of palmitic acid.     

Natural variation of GmFATA1B regulates seed oil content and composition in soybean

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl–acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

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.     

Quantitative trait locus analysis of saturated fatty acids in a population of recombinant inbred lines of soybean

Soybean [Glycine max (L.) Merr.] is an important crop which contributes approximately 58% of the world??s oilseed production. Palmitic and stearic acids are the two main saturated fatty acids in soybean oil. Different levels of saturated fatty acids are desired depending on the uses of the soybean oil. Vegetable oil low in saturated fatty acids is preferred for human consumption, while for industrial applications, soybean oil with higher levels of saturated fatty acids is more suitable. The objectives of this study were to identify quantitative trait loci (QTL) for saturated fatty acids, analyze the genetic effects of single QTL and QTL combinations, and discuss the potential of marker-assisted selection in soybean breeding for modified saturated fatty acid profiles. A population of recombinant inbred lines derived from the cross of SD02-4-59?×?A02-381100 was grown in five environments and the seed samples from each environment were evaluated for fatty acid content. Genotyping of the population was performed with 516 polymorphic single nucleotide polymorphism markers and 298 polymorphic simple sequence repeat markers. Eight QTL for palmitic acid, five QTL for stearic acid and nine QTL for total saturated fatty acids were detected by composite interval mapping and/or interval mapping, with a high level of consistency or repeatability in multiple environments. Most of these QTL have not been reported previously, with the exception of qPAL-A1 which confirmed the result of a previous study. Significant QTL?×?QTL interactions were not detected. However, significant QTL?×?environment interactions were detected in most cases. Comparisons of two-locus and three-locus combinations indicated that cumulative effects of QTL were significant for both palmitic and stearic acids. QTL pyramiding by molecular marker-assisted selection would be an appropriate strategy for improvement of saturated fatty acids in soybean.     

Evaluation of new Lesquerella and Physaria (Brassicaceae) oilseed germplasm

The seed oil of Lesquerella and the closely related genus Physaria (Brassicaceae) is rich in hydroxy fatty acids (HFAs). HFAs and their derivatives are used to produce a variety of industrial products including lubricants, nylon-11, plastics, drying agents, protective coatings, surfactants, cosmetics, and pharmaceuticals. Lesquerella fendleri is being developed as a new crop for arid regions of the southwestern United States as an alternative source of HFAs. Between 1995 and 2001, 66 accessions from 28 species of Lesquerella were collected in the United States, 33 accessions from four species were collected in Mexico, and 41 accessions from 15 species of Physaria were collected from the southwestern United States. Mean seed mass ranged from 0.54 to 2.30 mg for Lesquerella compared to 1.70 to 5.80 mg for Physaria. Seed oil content ranged from a high of 32.2% in Lesquerella to a high of 35.4% in Physaria. The fatty acid profile of all species of Physaria and most of the lesquerolic-acid-rich species of Lesquerella contained from 30 to 55% lesquerolic acid, although several species contained >60%. These collections of wild germplasm provide a diverse gene pool that should enhance our breeding program in developing a domestic source of HFAs.     

Lipids in Cruciferae

The effect of nitrogen, phosphorus and potassium nutrition on average seed weight, oil content and fatty acid composition of rape seed (Brassica napus) grown in soil-free culture has been studied. Nitrogen effected an increase in seed weight and a decrease in oil content, while the average amount of oil per seed remained constant. A small, but highly significant, decrease in palmitic and eicosenoic acid content, a significant decrease in oleic acid and a highly significant increase in erucic acid content were observed. This suggests that a decrease in the extent of elongation of oleic acid to erucic acid occurs in seeds developing on plants with sub-optimal levels of nitrogen nutrition. Phosphorus and potassium had very limited effects on fatty acid composition. Significant differences were found only in oleic acid content for phosphorus alone, the nitrogen-phosphorus interaction and the phosphorus-potassium interaction. The effect of various levels of sulfate at optimal levels for nitrogen, phosphorus and potassium, was studied in a separate experiment. Seed from sulfur-starved plants had decreased oil content; oleic acid percentages were increased and erucic acid percentages decreased. Excessive amounts of sulfate had no effect on fatty acid composition.     

Punicic acid production in Brassica napus

Punicic acid (PuA; 18:3Δ^{9cis,11trans,13cis}), a conjugated linolenic acid isomer bearing three conjugated double bonds, is associated with various health benefits and has potential for industrial use. The major nature source of this unusual fatty acid is pomegranate (Punica granatum) seed oil, which contains up to 80% (w/w) of its fatty acids as PuA. Pomegranate seed oil, however, is low yielding with unstable production and thus limits the supply of PuA. Metabolic engineering of established temperate oil crops for PuA production, therefore, has the potential to be a feasible strategy to overcome the limitations associated with sourcing PuA from pomegranate. In this study, the cDNAs encoding a pomegranate fatty acid conjugase and a pomegranate oleate desaturase were co-expressed in canola-type Brassica napus. Transgenic B. napus lines accumulated up to 11% (w/w) of the total fatty acids as PuA in the seed oil, which is the highest level of PuA reported in metabolically engineered oilseed crops so far. Levels of seed oil PuA were stable over two generations and had no negative effects on seed germination. The transgenic B. napus lines with the highest PuA levels contained multiple transgene insertions and the PuA content of B. napus seed oil was correlated with efficiency of oleic acid desaturation and linoleic acid conjugation. In addition, PuA accumulated at lower levels in polar lipids (5.0–6.9%) than triacylglycerol (7.5–10.6%), and more than 60% of triacylglycerol-associated PuA was present at the sn-2 position. This study provides the basis for the commercial production of PuA in transgenic oilseed crops and thus would open new prospects for the application of this unusual fatty acid in health and industry.