Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass

Abstract

SummaryWhen grown for energy production instead for smoking, tobacco can generate a large amount of inexpensive biomass more efficiently than almost any other agricultural crop. Tobacco possesses potent oil biosynthesis machinery and can accumulate up to 40% of seed weight in oil. In this work, we explored two metabolic engineering approaches to enhance the oil content in tobacco green tissues for potential biofuel production. First, an Arabidopsis thaliana gene diacylglycerol acyltransferase

AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum

“…We base this conclusion on the following lines of evidence: (i) TAG levels are reduced in abca9 and increased in ABCA9-overexpressing seeds; (ii) ABCA9 is expressed specifically in seeds at the middle and late stages of maturation, when storage lipids are rapidly accumulated and the rate of TAG synthesis is greatest (17); (iii) the temporal profile of ABCA9 transcript accumulation is closely correlated with the manifestation of abnormal seeds in developing abca9 siliques; (iv) TAG synthesis is reduced in abca9 seeds, as demonstrated by assimilation experiments with 14 C-acetate, 14 C-oleoyl-CoA, and 14 C-oleic acid; (v) ABCA9 is localized at the ER and belongs to a subfamily of lipid transporters, ABCA; and (vi) the relative proportions of fatty acids in TAGs are similar in WT and abca9 seeds, suggesting that the desaturation of esterified fatty acids on the glycerol backbone is not affected in abca9 and in turn, that only the first step at the ER, but not the later steps of lipid metabolism, is altered in the mutant (18)(19)(20)(21). If any steps further downstream in the TAG biosynthesis pathway were defective, then a marked alteration in the fatty acid composition of TAGs would be expected, because the ER is the site of extensive desaturation of fatty acids.…”

Compartmentation of Triacylglycerol Accumulation in Plants

“…Two classes of genes encode DGATs in plants, DGAT1 and DGAT2, and these are homologous to those found in fungi and mammals (42). Genetic studies with mutants have confirmed that DGAT1 is required for normal TAG accumulation in oilstoring tissues of Arabidopsis (43), and its overexpression in vegetative tissues or seeds can lead to enhanced TAG accumulation (44,45). However, disruption of DGAT1 gene function results in only a 20 -40% reduction in Arabidopsis seed oil, so other mechanisms must cooperate in the accumulation of TAG in plants.…”

ABI4 Activates DGAT1 Expression in Arabidopsis Seedlings during Nitrogen Deficiency

“…For instance, DGAT1 is up-regulated in senescing leaves, correlating with the plastid fatty acid partition into TAG (Kaup et al, 2002). Leaf-specific expression of DGAT1 in transgenic tobacco (Nicotiana tabacum) resulted in a 20-fold increase in TAG accumulation in leaves, and the total fatty acids also increased 2-fold up to 5.8% dry weight (Andrianov et al, 2010). In addition to DGAT1, PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1), which catalyzes the acyl-CoA-independent synthesis of TAG, also contributes to seed oil biosynthesis in Arabidopsis .…”

“…DGAT1, which catalyzes the final acylation step of sn-1,2-diacylglycerol to TAG, is thought to be the ratelimiting enzyme of TAG biosynthesis (Ichihara et al, 1988). Arabidopsis dgat1 mutant seeds only accumulate about 55% to 75% of TAG (Routaboul et al, 1999;Zou et al, 1999;Kaup et al, 2002), whereas seedspecific overexpression of DGAT1 increases oil content from 11% to 28% (Jako et al, 2001;Taylor et al, 2009;Andrianov et al, 2010). DGAT1 is also important for TAG accumulation in leaves (Slocombe et al, 2009).…”