Dedicated Industrial Oilseed Crops as Metabolic Engineering Platforms for Sustainable Industrial Feedstock Production.

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil.

Seed oil composed of wax esters with long-chain monoenoic acyl moieties represents a high-value commodity for industry. Such plant-derived sperm oil-like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low-input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl-CoA substrates. To produce plant-derived sperm oil-like liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1∆c/Oleo3:EYFP:MmWS, Oleo3:mCherry:MmFAR1∆c/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.

Production of wax esters in plant seed oils by oleosomal cotargeting of biosynthetic enzymes.

Wax esters are neutral lipids exhibiting desirable properties for lubrication. Natural sources have traditionally been whales. Additionally some plants produce wax esters in their seed oil. Currently there is no biological source available for long chain length monounsaturated wax esters that are most suited for industrial applications. This study aimed to identify enzymatic requirements enabling their production in oilseed plants. Wax esters are generated by the action of fatty acyl-CoA reductase (FAR), generating fatty alcohols and wax synthases (WS) that esterify fatty alcohols and acyl-CoAs to wax esters. Based on their substrate preference, a FAR and a WS from Mus musculus were selected for this study (MmFAR1 and MmWS). MmWS resides in the endoplasmic reticulum (ER), whereas MmFAR1 associates with peroxisomes. The elimination of a targeting signal and the fusion to an oil body protein yielded variants of MmFAR1 and MmWS that were cotargeted and enabled wax ester production when coexpressed in yeast or Arabidopsis. In the fae1 fad2 double mutant, rich in oleate, the cotargeted variants of MmFAR1 and MmWS enabled formation of wax esters containing >65% oleyl-oleate. The data suggest that cotargeting of unusual biosynthetic enzymes can result in functional interplay of heterologous partners in transgenic plants.

Homo- and heterodimers of tobacco bZIP proteins counteract as positive or negative regulators of transcription during pollen development.

Expression of BZI-1 Delta N, a dominant-negative form of the tobacco (Nicotiana tabacum) basic leucine zipper (bZIP) transcription factor BZI-1 leads to severe defects in pollen development which coincides with reduced transcript abundance of the stamen specific invertase gene NIN88 and decreased extracellular invertase enzymatic activity. This finding suggests a function of BZI-1 in regulating carbohydrate supply of the developing pollen. BZI-1 heterodimerises with the bZIP factors BZI-2, BZI-3 and BZI-4 in vitro and in planta. Whereas BZI-1 exhibits only weak activation properties, BZI-1/BZI-2 heterodimers strongly activate transcription. Consistently, approaches leading to reduced levels of functional BZI-1 or BZI-2 both significantly interfere with pollen development, auxin responsiveness and carbohydrate partitioning. In situ hybridisation studies for BZI-1 and BZI-2 confirmed temporal and spatial overlapping expression patterns in tapetum and pollen supporting functional cooperation of these factors during pollen development. Plants over-expressing BZI-4 produce significantly reduced amounts of intact pollen and are also impaired in NIN88 transcription and enzymatic activity. BZI-4 homodimer efficiently binds to a G-box located in the NIN88 promoter but exhibits almost no transcriptional activation capacity. As BZI-4 does not actively repress transcription, we propose that its homodimer blocks G-box mediated transcription. In summary, these data support a regulatory model in which BZI-4 homodimers and BZI-1/BZI-2 heterodimers perform opposing functions as negative or positive transcriptional regulators during pollen development.