Systematic Metabolic Engineering Of Palmitoleic Acid In Soybean And Tobacco

Palmitoleic acid (16:1Δ9) is an unusual monounsaturated fatty acid consisting of 16 carbon elements and a double bond located at the ninth carbon position counting from the carboxyl end (e.g. the seventh carbon position counting from the CH3 end). This fatty acid is highly valued for human nutrition, medication and industry. Recently, there has been considerable interest in using 16:1Δ9 in foods and nutriceuticals for health purposes. Accumulating studies indicate that palmitoleic acid can increase cell membrane fluidity, inhibit oncogenesis, reduce inflammation associated with diabetes, heart disease and other health problems. However, its precursor palmitic acid (16:0) composed of 16 carbons and no double bond, the major portion of the saturated fatty acids in most vegetable oil, functions negatively in these regards. Moreover, palmitoleate is recognized as a good renewable source of 1-octene, a high-demand feedstock used as a co-monomer in the expanding production of linear low density polyethylene. Plant oils rich in palmitoleic acid are the ideal resource for biodiesel production since 16:1Δ9 exhibits highly antioxidant quality and cold resistance.A number of natural wild plants can synthesize high levels of the fatty acid in seeds, but low yields and poor agronomic properties of those plants preclude their commercial use for production. However, a very tiny amount of 16:1Δ9 was present in common oilseeds such as soybean (Glycine max) and rape seed (Brassica napus). In this study, therefore, an integrate approach consisting of genomics, molecular biology and gene engineering tools was employed to investigate the mechanism of palmitoleic acid biosynthesis and regulation in plant tissues. Another aim is to assembly the biosynthesis pathway of palmitoleic acid in seeds of soybean, a typical common oilseed and vegetable tissues of tobacco, the model plant with high biomass so as to develop novel germplasm of soybean and tobacco plant with high accumulation of palmitoleic acid in seed and vegetable organs, respectively. Such new type of soybean oil is much valued for human health, and thus suitable for functional food production. In addition, the oil is desirable material for production of high-quality biodiesel. Tobacco germplasm with high accumulation of palmitoleic acid in vegetable tissues/organs could be further used in breeding of new varieties special for biodiesel production. Commercialization of such variety can enhance economic efficiency of tobacco cultivation and speed up the production of renewable "green energy" without affecting food security unlike staple crops used for biodiesel production.A number of new findings are obtained from the present study. Of them, four main findings are summarized as the followings:1. Palmitoleic acid (16:1A9) is synthesized from palmitic acid (16:0) catalyzed by acyl-Δ 9 desaturases. Acyl-A9 desaturases are the key enzymes needed for metabolic assembly of palmitoleic acid biosynthetic pathway in soybean and tobacco. Two cDNA clones of acyl-CoA-Δ9 desaturases, namely ScCoA-A9D (1533 bp) and PoCoA-A9D (1272 bp), were isolated from yeast (Saccharomyees cerevisiae) and oyster mushroom (Pleurotus ostreatus), respectively. It is the first time to get the experimental evidence that ScCoA-A9D and PoCoA-A9D have high substrate specificity prefer to 16:0 by auxotrophic yeast expression system. Further mutation analysis showed that one of the key sites of enzyme activity was a conserved glycine located at the 309th amino acid residue (G309) of ScCoA-A9D and at the 237 amino acid residue (G237) of PoCoA-A9D. Instant expression by agro-infiltration on petunia leaves revealed that the two fungus cytosolic CoA-A9 desaturases could correctly function in the cells and tissues of high plants to catalyze CoA-16:0 to form CoA-16:1Δ9 on endosome reticulum (ER). The two cDNAs are the target genes for assembling palmitoleic acid biosynthesis pathway on ER in cytoplasm.2. Acyl-ACP-A9 desaturase (ACP-A9D) is another type of acyl-Δ9 desaturase which catalyze ACP-16:0 to form ACP-16:1A9 in plastid. A cDNA clone (1188 bp) encoding plastidal acyl-ACP-Δ9D ( MucACP-A9D) was isolated from developing seeds of cat’s claw (Macfadyena unguis-cati), a high natural accumulator of 16:1A9. For functional analysis of MucACP-A9D, several experimental tools were employed, which include E. coli expression, target enzyme purification, isotope labeling substrate and in vitro essay. The examination evidenced a high specificity of MucACP-A9D to substrate 16:0-ACP. The cDNA of Muc A9D can be used to assembly palmitoleic acid synthesis pathway in chloroplast/plastid for increasing palmitoleic acid level in plant tissues. 3. A yeast (Saccharomyees cerevisiae) acyl-CoA-A9 desaturase (ScCoA-A9D, shorted as ScA9D) was expressed by subcellular targeting in soybean (Glycine max) seeds with the goal of increasing palmitoleic acid level and simultaneously decreasing the saturated fatty acid content in the seed oil. Seed-specific expression vector of ScΔ9D was constructed, and further used for soybean transformation by soybean somatic embryo-based biolistic particle bombardment. A number of transgenic soybean plants were generated, and consequently matured transgenic seeds were harvested. The mRNA expression,Western blotting, and seed lipid analysis by gas chromatograph demonstrated that ScA9D was correctly expressed and worked in ER and plastid, respectively. The yeast A9 desaturase expression resulted in the conversion of palmitic acid (16:0) to palmitoleic acid (16:1A9) in soybean seeds compared to the wild-type and vector control plants. Moreover, higher levels of 16:1A9 and its elongation product ds-vaccenic acid (18:1Δ11) were produced by the plastid-targeted expression of the enzyme, indicating that yeast cytosolic acyl-CoA-Δ9 desaturase works more effectively in the plastid. Other alternations in lipid metabolism include the reduction of polyunsaturated fatty acids (18:2 and 18:3) and the overall saturated fatty acid content, suggesting that a mechanism exists further downstream in oil biosynthesis to compensate the fatty acid alternation. This is the first time a cytosolic acyl-CoA-Δ9 desaturase has been functionally expressed in chloroplast/plastid and the stronger activity was achieved than its cytosolic expression. Thus, the present study provides a new strategy for converting 16:0 to 16:1Δ9 by protein engineering of acyl-CoA-A9 desaturase in commercialized oil crops.4. To increase accumulation of palmitoleic acid in plant vegetable tissues/organs, a yeast (Saccharomyees cerevisiae) acyl-CoA-A9 desaturase (ScA9D) was used for cytosol-and plastid-targeting expression in tobacco (Nicotiana tabacum L.). Another aim is to investigate the effects of the subcellular-targeted expression of this enzyme on lipid synthesis and metabolism in plant system. A constitutive expression vector of ScA9D was developed and further used for tobacco transformation by agrobacterium-mediated method. Compared to the wild type and vector control plants, the contents of monounsaturated palmitoleic (16:1Δ9) and cis-vaccenic (18:1Δ11) were significantly enhanced in the ScΔ9D-transgenic leaves whereas the levels of saturated palmitic acid (16:0) and polyunsaturated linoleic (18:2) and linolenic (18:3) acids were reduced in the transgenics. Notably, the contents of 16:1Δ9 and 18:1Δ11 in the ScA9D plastidal-expressed leaves were 2.7 and 1.9 folds of that in the cytosolic-expressed tissues. Statistical analysis appeared a negative correlation coefficient between 16:0 and 16:1A9 levels. The present data indicate that yeast cytosolic acyl-CoA-Δ9 desaturase can convert palmitic (16:0) into palmitoleic acid (16:1A9) in high plant cells. Moreover, this effect of the enzyme is stronger with the plastid-targeted expression than the cytosol-target expression. A new strategy was developed for high accumulation of ω-7 fatty acids (16:1A9 andl8:1Δ11) in plant tissues by protein engineering of acyl-CoA-A9 desaturase. The findings would particularly benefit the metabolic assembly of the lipid biosynthesis pathway in the large-biomass vegetative organs such as tobacco leaves for the production of high-quality biodiesel.The present findings will benefit understanding of seed oil biosynthesis, particularly unusual fatty acid synthesis pathway and its regulation in plant system.In addition, the data provide new gene elements and technology tools for efficient assembly of oil biosynthesis pathway and oil nutrition improvement in oilseeds such as soybean and other high biomass crops like tobacco. Such knowledge and techniques developed in this study will also expand industrial implications of the lipid renewable resources.