Metabolic Engineering Plant Seeds with Fish Oil-Like Levels of DHA

Background

Omega-3 long-chain (≥C₂₀) 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 C₂₀ 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 C₂₂ 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 C₂₂ fatty acid DHA in Arabidopsis thaliana seed oil with a high ω3/ω6 ratio. This was achieved using a transgenic pathway to increase the C₁₈ 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 a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds

Camelina sativa (camelina) is emerging as an alternative oilseed crop due to its short growing cycle, low input requirements, adaptability to less favorable growing environments and a seed oil profile suitable for biofuel and industrial applications. Camelina meal and oil are also registered for use in animal and fish feeds; however, like meals derived from most cereals and oilseeds, it is deficient in certain essential amino acids, such as lysine. In higher plants, the reaction catalyzed by dihydrodipicolinate synthase (DHDPS) is the first committed step in the biosynthesis of lysine and is subject to regulation by lysine through feedback inhibition. Here, we report enhancement of lysine content in C. sativa seed via expression of a feedback inhibition-insensitive form of DHDPS from Corynebacterium glutamicums (CgDHDPS). Two genes encoding C. sativa DHDPS were identified and the endogenous enzyme is partially insensitive to lysine inhibition. Site-directed mutagenesis was used to examine the impact of alterations, alone and in combination, present in lysine-desensitized DHDPS isoforms from Arabidopsis thaliana DHDPS (W53R), Nicotiana tabacum (N80I) and Zea mays (E84K) on C. sativa DHDPS lysine sensitivity. When introduced alone, each of the alterations decreased sensitivity to lysine; however, enzyme specific activity was also affected. There was evidence of molecular or structural interplay between residues within the C. sativa DHDPS allosteric site as coupling of the W53R mutation with the N80V mutation decreased lysine sensitivity of the latter, but not to the level with the W53R mutation alone. Furthermore, the activity and lysine sensitivity of the triple mutant (W53R/N80V/E84T) was similar to the W53R mutation alone or the C. glutamicum DHDPS. The most active and most lysine-insensitive C. sativa DHDPS variant (W53R) was not inhibited by free lysine up to 1 mM, comparable to the C. glutamicums enzyme. Seed lysine content increased 13.6 -22.6% in CgDHDPS transgenic lines and 7.6–13.2% in the mCsDHDPS lines. The high lysine-accumulating lines from this work may be used to produce superior quality animal feed with improved essential amino acid profile.

     

Sequencing of Camelina neglecta,a diploid progenitor of the hexaploid oilseed Camelina sativa

Camelina neglecta is a diploid species from the genus Camelina, which includes the versatile oilseed Camelina sativa. These species are closely related to Arabidopsis thaliana and the economically important Brassica crop species, making this genus a useful platform to dissect traits of agronomic importance while providing a tool to study the evolution of polyploids. A highly contiguous chromosome-level genome sequence of C. neglecta with an N50 size of 29.1 Mb was generated utilizing Pacific Biosciences (PacBio, Menlo Park, CA) long-read sequencing followed by chromosome conformation phasing. Comparison of the genome with that of C. sativa shows remarkable coincidence with subgenome 1 of the hexaploid, with only one major chromosomal rearrangement separating the two. Synonymous substitution rate analysis of the predicted 34 061 genes suggested subgenome 1 of C. sativa directly descended from C. neglecta around 1.2 mya. Higher functional divergence of genes in the hexaploid as evidenced by the greater number of unique orthogroups, and differential composition of resistant gene analogs, might suggest an immediate adaptation strategy after genome merger. The absence of genome bias in gene fractionation among the subgenomes of C. sativa in comparison with C. neglecta, and the complete lack of fractionation of meiosis-specific genes attests to the neopolyploid status of C. sativa. The assembled genome will provide a tool to further study genome evolution processes in the Camelina genus and potentially allow for the identification and exploitation of novel variation for Camelina crop improvement.     

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - IBA Indole-3-butyric acid - GA₃ gibberellic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) basal medium     

Genetic mapping of agronomic traits in false flax (Camelina sativa subsp. sativa).

The crucifer oilseed plant false flax (Camelina sativa subsp. sativa) possesses numerous valuable agronomic attributes that make it attractive as an alternative spring-sown crop for tight crop rotations. The oil of false flax is particularly rich in polyunsaturated C18-fatty acids, making it a valuable renewable feedstock for the oleochemical industry. Because of the minimal interest in the crop throughout the 20th century, breeding efforts have been limited. In this study, a genetic map for C. sativa was constructed, using amplified fragment length polymorphism (AFLP) markers, in a population of recombinant inbred lines that were developed, through single-seed descent, from a cross between 'Lindo' and 'Licalla', 2 phenotypically distinct parental varieties. Three Brassica simple sequence repeat (SSR) markers were also integrated into the map, and 1 of these shows linkage to oil-content loci in both C. sativa and Brassica napus. Fifty-five other SSR primer combinations showed monomorphic amplification products, indicating partial genome homoeology with the Brassica species. Using data from field trials with different fertilization treatments (0 and 80 kg N/ha) at multiple locations over 3 years, the map was used to localize quantitative trait loci (QTLs) for seed yield, oil content, 1000-seed mass, and plant height. Some yield QTLs were found only with the N0 treatment, and might represent loci contributing to the competitiveness of false flax in low-nutrient soils. The results represent a starting point for future marker-assisted breeding.     

Phytotoxicity of green synthesized silver nanoparticles on Camelina sativa L

Due to the increased production and release of silver nanoparticles (AgNPs) in the environment, the concerns about the possibility of toxicity and oxidative damage to plant ecosystems should be considered. In the present study, the effects of different concentrations of AgNPs (0, 0.5, 1, 2, 3 and 4 g/L) synthesized using the extract of camelina (Camelina sativa) leaves on the growth and the biochemical traits of camelina seedlings were investigated. The results showed that AgNPs significantly increased Ag accumulation in the roots and shoots which decreased the growth and photosynthetic pigments of camelina seedlings. The highest decrease in the height and total dry weight was observed by 53.1 and 61.8% under 4 g/L AgNPs, respectively over control plants. AgNPs application over 2 g/L enhanced the accumulation of proline, malondialdehyde, hydrogen peroxide and methylglyoxal, and up-regulated the activity of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) and glyoxalase (glyoxalase I and II) system which indicates oxidative stress induction in camelina seedlings. Moreover, AgNPs reduced ASA and GSH contents and increased DHA and GSSG contents, hence disrupting the redox balance. These results showed that AgNPs at 4 g/L had the most toxic effects on the camelina growth. Therefore, increasing oxidative stress markers and the activity of antioxidant enzymes and enzymes involved in glyoxalase system indicated the oxidative stress induced by AgNPs treatments over 2 g/L as well as the induction of antioxidant defense systems to combat AgNPs-induced oxidative stress.     

亚麻荠种植和利用的研究现状

介绍了亚麻荠(Camelina sativa (L.) Crantz)的种植历史、栽培特性、种子成分、生物学特性、栽培措施、抗病虫害能力以及对其产物的应用。亚麻荠耐旱和抗病虫草能力强的特性符合农业可持续发展的方向。作为一种低投入的经济作物,亚麻荠的种植在我国有着广阔的发展前景。     

亚麻荠种植和利用的研究现状

介绍了亚麻荠(Camelina sativa(L.)Crantz)的种植历史、栽培特性、种子成分、生物学特性、栽培措施、抗病虫害能力以及对其产物的应用.亚麻荠耐旱和抗病虫草能力强的特性符合农业可持续发展的方向.作为一种低投入的经济作物,亚麻荠的种植在我国有着广阔的发展前景.     

Resistance to Rhizoctonia solani and Presence of Antimicrobial Compounds in Camelina sativa Roots

Camelina sativa (L.) Crantz was significantly more resistant to Rhizoctonia solani Kühn than [itBrassica napus L. cv Westar. Emergence of C. sativa seedlings was 22 to 33% greater than those of Westar in R. solani-infested soil. The greater resistance of C. sativa seedlings to R. solani appeared to be due to greater amounts of antimicrobial compounds present in C. sativa roots. These antimicrobial compounds inhibited the growth of both weakly virulent and virulent R. solani] isolates to the same extent. Four antimicrobial compounds were purified from C. sativa roots and their structures elucidated. Two were identified as the phytoalexins (camalexin and methoxycamalexin) previously described from C. sativa leaves. This appears to be the first report of elicitation of phytoalexins from roots of crucifers. Two preformed antimicrobial compounds were identified as methyl 1-methylindole-3-carboxylate and 10-methyl sulfinyldecylisothiocyanate.     

代谢工程

代谢工程,也称途径工程,是基因工程一个重要分支,一般是多基因的基因工程,与细胞的基因调控、代谢调控和生化工程密切相关。讨论了代谢工程的应用,包括通过改变代谢流和代谢途径提高产量,改善生产过程,构建新的代谢途径和产生新的代谢产物等。     

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.     

Imaging heterogeneity of membrane and storage lipids in transgenic Camelina sativa seeds with altered fatty acid profiles

Engineering compositional changes in oilseeds is typically accomplished by introducing new enzymatic step(s) and/or by blocking or enhancing an existing enzymatic step(s) in a seed‐specific manner. However, in practice, the amounts of lipid species that accumulate in seeds are often different from what one would predict from enzyme expression levels, and these incongruences may be rooted in an incomplete understanding of the regulation of seed lipid metabolism at the cellular/tissue level. Here we show by mass spectrometry imaging approaches that triacylglycerols and their phospholipid precursors are distributed differently within cotyledons and the hypocotyl/radicle axis in embryos of the oilseed crop Camelina sativa, indicating tissue‐specific heterogeneity in triacylglycerol metabolism. Phosphatidylcholines and triacylglycerols enriched in linoleic acid (C18:2) were preferentially localized to the axis tissues, whereas lipid classes enriched in gadoleic acid (C20:1) were preferentially localized to the cotyledons. Manipulation of seed lipid compositions by heterologous over‐expression of an acyl–acyl carrier protein thioesterase, or by suppression of fatty acid desaturases and elongases, resulted in new overall seed storage lipid compositions with altered patterns of distribution of phospholipid and triacylglycerol in transgenic embryos. Our results reveal previously unknown differences in acyl lipid distribution in Camelina embryos, and suggest that this spatial heterogeneity may or may not be able to be changed effectively in transgenic seeds depending upon the targeted enzyme(s)/pathway(s). Further, these studies point to the importance of resolving the location of metabolites in addition to their quantities within plant tissues.     

Genome-wide comparative analysis of Mg transporter gene family between Triticum turgidum and Camelina sativa

Magnesium (Mg) as a bimetal plays critical roles in biochemical processes, membrane stability, and enzyme activity. Mg transporters (MGTs) are involving in maintaining Mg homeostasis in cells. Although the MGT family members have been identified in different plant species, there is no comprehensive analysis of the other plants' MGT genes. In the current study, 62 and 41 non-redundant putative MGT proteins were recognized into the genome of Camelina sativa, and Triticum turgidum and they were compared based on physicochemical properties, protein structure, expression, and interaction. All identified MGTs were classified into three subgroups, NIPA, CorA, and MRS2/MGT, based on conserved-motifs distribution. The results showed that the secondary structure pattern in NIPA and MRS2 subfamily members in both studied plant species were highly similar. Furthermore, MGTs encompass the conserved structures and the critical sites mainly in the metal ion and Mg²⁺ binding centers as well as the catalytic sites were observed. The highest numbers of protein channels were predicted in CorA proteins in both C. sativa and T. turgidum with 24 and 17 channel numbers, respectively. The Ser, Pro, Gly, Lys, Tyr, and Arg amino acids were predicted as the binding residues in MGTs channel regions. The expression pattern of identified genes demonstrated that MGT genes have diverse tissue-specific expression and stress response expression patterns. Besides, 147 co-expressed genes with MGTs were clustered into the eight co-expression nodes involved in N-glycan biosynthesis, protein processing in the endoplasmic reticulum, carbon metabolism, biosynthesis of amino acids, and endocytosis. In the present study, all interpretations are based on in silico predictions, which can be used in further studies related to functional genomics of MGT genes.