Plant genetic engineering has been considered to be a solution for such global challenges. Molecular biology technology using synthetic or modified genes with appropriate sensible genetic engineering strategies can help to increasing or create a new product. This potentially would lead to high production yields at low costs of better quality oils which are important for green energy and industrial materials.
Redirection of Metabolic Flux for High Levels of Omega-7 Monounsaturated Fatty Acid Accumulation...
Written by nguyen
Monday, 05 May 2014 16:23
Plant Biotechnology Journal.
First published: 26 July 2014
Authors: Huu Tam Nguyen1, Hyunwoo Park2, Karen L. Koster3, Rebecca E. Cahoon1, Hanh TM Nguyen2, John Shanklin4, Thomas E. Clemente2, and Edgar B. Cahoon*1
Seed oils enriched in omega-7 monounsaturated fatty acids, including palmitoleic acid (16:1D9) and cis-vaccenic acid (18:1D11), 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 delta 9 desaturation of stearoyl (18:0)-acyl carrier protein (ACP) to delta 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 delta 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 monounsaturated fatty acid accumulation to ~ 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 delta 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 non-transformed 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.
Camelina Seed Transcriptome: A Tool for Meal and Oil Improvement and Translational Research
Written by nguyen
Wednesday, 03 October 2012 23:02
Plant Biotechnology Journal
Authors: Tam Nguyen, Jillian E. Silva, Ram Podicheti, Jason Macrander, Wenyu Yang, Tara J. Nazarenus, Jeong-Wan Nam, Jan G. Jaworski, Chaofu Lu, Keithanne Mockaitis, Edgar B. Cahoon.
Abstract: Camelina (Camelina sativa), a Brassicaceae oilseed, has received intense interest as a biofuel crop and production platform for industrial oils. Limiting wider production of camelina for these uses is the need to improve the quality and content of the seed protein rich-meal and oil, which is enriched in oxidatively unstable polyunsaturated fatty acids that are deleterious for biodiesel. To identify candidate genes for meal and oil quality improvement, we built a transcriptome reference from 2,047 Sanger ESTs and over 2 million 454-derived sequence reads, representing genes expressed in developing camelina seeds. The transcriptome of ~60K transcripts from 22,597 putative genes includes camelina homologs of nearly all known seed-expressed genes, suggesting a high level of completeness and usefulness of the reference. These sequences included candidates for 12S (cruciferins) and 2S (napins) seed storage proteins (SSPs) and nearly all known lipid genes, which have been compiled into an accessible database. To demonstrate the utility of the transcriptome for seed quality modification, seed-specific RNAi lines deficient in napins were generated by targeting 2S SSP genes, and high oleic acid oil lines were obtained by targeting fatty acid desaturase 2 (FAD2) and fatty acid elongase 1 (FAE1). The high sequence identity between Arabidopsis and camelina genes was also exploited to engineer high oleic lines by RNAi with Arabidopsis FAD2 and FAE1 sequences. It is expected that this transcriptomic data will be useful for breeding and engineering of additional camelina seed traits and for translating findings from the model Arabidopsis thaliana to an oilseed crop.
Authors:Huu Tam Nguyen, Girish Mishra, Edward Whittle, Scott A. Bevan, Ann Owens Merlo, Terence A. Walsh and John Shanklin
Financial Source:This work was supported by the Office of Basic Energy Sciences of the US Department of Energy, and The Dow Chemical Company and Dow AgroSciences.
Plant oils containing omega-7 fatty acids (FA) (palmitoleic 16:1 delta9 and cis-vaccenic 18:1delta11) have potential as sustainable feedstocks for producing industrially important octene via metathesis chemistry. Engineering plants to produce seeds that accumulate high levels of any unusual FA has been an elusive goal. We have achieved high levels of omega-7 FA accumulation by systematic metabolic engineering of Arabidopsis thaliana. A plastidial 16:0-ACP desaturase has been engineered to convert 16:0 to 16:1?9 with specificity >100-fold that that of naturally-occurring paralogs such as that from Doxantha unguis-cati L. Expressing this engineered enzyme (Com25) in seeds increased omega-7 FA accumulation from <2% to 14%. Reducing competition for 16:0-ACP by downregulating the KASII 16:0 elongase further increased accumulation of omega-7 FA to 56%. The level of 16:0 exiting the plastid without desaturation also increased to 21%. Coexpression of a pair of fungal 16:0 desaturases in the cytosol reduced the 16:0 level to 11% and increased omega-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.
There is increasing interest in the use of plant oils as renewable sources of industrial chemical feedstocks (Dyer et al., 2008; Carlsson, 2009). Recent developments in olefin metathesis have demonstrated that long chain monoene FA from vegetable oils can be efficiently split into the corresponding short chain alpha olefin and ?-alkenoic acids (Rybak et al., 2008; Meier, 2009). Thus ethenolytic metathesis of ?-7 FA such as palmitoleic or cis-vaccenic acids yields 1-octene and 9-decenoate. 1-Octene is a high-demand feedstock with a global consumption of over half a million tonnes per year that is primarily used as a comonomer in the expanding production of linear low density polyethylene. It is mainly synthesized from petroleum-derived ethylene via oligomerization to yield a complex mixture of alpha olefins, or from coal-derived syngas (Systems, 2007).
Altering Arabidopsis Oilseed Composition by a Combined. Antisense-Hairpin RNAi Gene Suppression Approach
Written by Tam Nguyen
Friday, 06 February 2009 05:00
Tam Nguyen1 and John Shanklin1
Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA
Received: 16 September 2008 Revised: 29 September 2008 Accepted: 3 November 2008 Published online: 5 December 2008
Abstract Antisense (AS) and hairpin (HP) RNA interference (RNAi) targeted gene suppression technologies have been used to modify seed oil composition. Larger numbers of AS transgenics have to be screened to achieve a targeted level of suppression compared to RNAi. We hypothesized combining AS with RNAi might result in enhanced gene suppression compared to either method individually. AS and HP-RNAi were combined as hairpinantisense (HPAS) constructs containing ~125 bp sense and antisense portions of an untranslated region of the target gene separated by an intron containing an antisense copy of a portion of the target coding region. The ?12-desaturase FAD2, the ?3-desaturase FAD3 and ?-ketoacyl-ACP synthase (KAS) II were targeted in Arabidopsis to evaluate changes in oil composition with AS, HP and HPAS constructs driven by the phaseolin promoter. Modest but statistically significant enhancements in oilseed phenotypes were observed with HPAS relative to AS and HP-RNAi. Phenotypes for HPAS suppression of FAD2 and FAD3 were indistinguishable from their strongest mutant alleles. Our data suggest that HPAS may be useful for: (1) achieving levels of suppression comparable to those of gene knockouts in a tissue specific manner. (2) Maximizing suppression of suboptimal RNAi constructs and (3) minimizing the screening of transgenics to achieve desired oilseed composition.
Mark S. Pidkowich*, Huu Tam Nguyen*, Ingo Heilmann, Till Ischebeck, and John Shanklin†
Department of Biology, Brookhaven National Laboratory, Upton, NY 11973
Edited by George H. Lorimer, University of Maryland, College Park, MD, and approved January 22, 2007 (received for review December 14, 2006)
beta-Ketoacyl-acyl carrier protein (ACP) synthase II (KASII) elongates 16:0-ACP to 18:0-ACP in the plastid, where it competes with three other enzymes at the first major branch point in fatty acid biosynthesis. Despite its key metabolic location, the influence of KASII in determining seed oil composition remains unclear, in part because the biochemical consequences of the fab1-1 mutation were unresolved. Thus, fab1-1, and a newly identified knockout allele, fab1-2, were analyzed in the context of the hypothesis that modulating KASII activity is sufficient to convert the composition of a temperate seed oil into that of a palm-like tropical oil. No homozygous fab1-2 individuals were identified in progeny of self-fertilized heterozygous fab1-2 plants, ?1/4 of which aborted before the torpedo stage, suggesting that fab1-2 represents a complete loss of function and results in lethality when homozygous. Consistent with this hypothesis, homozygous fab1-2 plants were identified when a fab1-1 transgene was introduced, demonstrating that fab1-1 encodes an active KASII. Strong seed-specific hairpin-RNAi reductions in FAB1 expression resulted in abortion of ?1/4 of the embryos in an apparent phenocopy of fab1-2 homozygosity. In less severe FAB1 hairpin-RNAi individuals, embryos developed normally and exhibited a 1:2:1 segregation ratio for palmitate accumulation. Thus, early embryo development appears sensitive to elevated 16:0, whereas at later stages, up to 53% of 16:0, i.e., a 7-fold increase over wild-type levels, is tolerated. These results resolve the role of KASII in seed metabolism and demonstrate that modulation of Arabidopsis KASII levels is sufficient to convert its temperate oilseed composition to that of a palm-like tropical oil.
(*) Mark S Pidkowich and Huu Tam Nguyen contributed equally to this work
A widely applicable bacteriophage T7 RNA polymerase-directed, tissue-specific and inducible over-expression of foreign genes in transgenic plants was developed. This was achieved through the simultaneous transformation of a modified T7 RNA polymerase to specifically transcribe the foreign gene placed under the control of T7 expression signals. The T7 RNA polymerase recognized the chimeric uidA gene integrated randomly into tobacco and rice genomes. Results from the use of six different promoters with different tissue specificities indicated that the recombinant protein was expressed at a several-fold (3–10-fold) higher level when compared with transgenes expressed directly under the control of these tissue-specific promoters. An important feature of the T7 system in plants was the near-uniform expression in the independently transformed plants, in contrast with the large variations observed in transgene expression under the direct control of plant promoters. In addition, our results demonstrated the application of the T7 system in the regulation of transgene expression through chemically inducible mechanisms. This versatility of controlled and regulated expression offers a powerful tool that could be used in various programmes in plant biotechnology and genomic studies.