Synthetic redesign of plant lipid metabolism

Abstract

Plant seed lipid metabolism is an area of intensive research, including many examples of transgenic events in which oil composition has been modified. In the selected examples described in this review, progress towards the predictive manipulation of metabolism and the reconstitution of desired traits in a non-native host is considered. The advantages of a particular oilseed crop, Camelina sativa, as a flexible and utilitarian chassis for advanced metabolic engineering and applied synthetic biology are considered, as are the issues that still represent gaps in our ability to predictably alter plant lipid biosynthesis. Opportunities to deliver useful bio-based products via transgenic plants are described, some of which represent the most complex genetic engineering in plants to date. Future prospects are considered, with a focus on the desire to transition to more (computationally) directed manipulations of metabolism.

Keywords: fatty acid metabolism; metabolic engineering; oil crops; plant biotechnology; predictive manipulation.

Figure 1

Schematic diagram of the major pathways involved in the production of triacylglycerol (TAG) in seeds. The successful synthesis of eicosopentanoic acid (EPA) and docosahexaenoic acid (DHA), avoiding metabolic bottlenecks, requires the coordinated use of coenzyme A (CoA)‐based desaturase and elongase activities. The incorporation of these long chain omega‐3 fatty acids into TAG then depends on the activities of endogenous TAG assembly pathways. Metabolite abbreviations: ACP, acyl carrier protein; DAG, diacylglycerol; G3P, glycerol‐3‐phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; PC phosphatidylcholine, TAG, triacylglycerol. Enzyme abbreviations: CPT, CDP‐choline:diacylglycerol cholinephosphotransferase; DGAT, diacylglycerol acyltransferase; FAD, fatty acid desaturase; FAS, type II fatty acid synthase complex; GPAT, glycerol‐3‐phosphate acyltransferase, KAS, β‐ketoacyl‐ACP synthase; LPAAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; PDCT, phosphatidylcholine:diacylglycerol cholinephosphotransferase; PLC, phospholipase C; SAD, Δ9 stearoyl‐acyl carrier protein desaturase.

Figure 2

Strategy for metabolic redesign of fatty acid desaturation and biosynthetic pathways for production of omega‐7 monounsaturated fatty acids. Seeds of most oil crops accumulate unsaturated fatty acids derived almost entirely from the omega‐9 desaturation pathway that begins in plastids with the Δ9 desaturation of the fatty acid synthase (FAS) product stearoyl (18:0)‐acyl carrier protein (ACP) desaturase. The rational design of a mutant Δ9 acyl‐ACP desaturase (‘Com25’) with specificity for palmitoyl (16:0)‐ACP enabled the redirection of fatty acid flux for the synthesis of the omega‐7 monounsaturated fatty acid palmitoleic acid (16:1Δ9) in Camelina seeds. Enhancement of 16:0‐ACP substrate pools for the Com25 Δ9 desaturase was increased by RNA interference (RNAi) suppression of genes for the FatB thioesterases that releases 16:0 from ACP and the β‐ketoacyl‐ACP synthase II (KASII) that elongates 16:0‐ACP to 18:0‐ACP. Caenorhabditis elegans FAT5 Δ9 desaturase was introduced to obtain additional production of 16:1Δ9 from 16:0‐CoA in the endoplasmic reticulum (ER). RNAi suppression of the gene for the fatty acid elongase 1 (FAE1) β‐ketoacyl‐CoA synthase was conducted to block fatty acid elongation and enhance accumulation of C16 and C18 omega‐7 monounsaturated fatty acids.

Figure 3

Strategy for redesign of seed storage oil synthesis for production of sn‐3 acetyl triacylglycerol (acTAG). Seeds of most oil crops accumulate TAGs generated, in part, through the activity of diacylglycerol (DAG) acyltransferase 1 (DGAT1) that uses DAG and a long‐chain fatty acyl‐CoA as substrates. Through the transgenic expression of a Euonymus alatus (burning bush) diacylglycerol acetyl‐CoA transferase (EaDAcT), seed storage oil can be shifted to the production of acTAG, rather than ‘regular’ TAG with long‐chain fatty acids (lcTAG). RNA interference suppression of the gene for DGAT1 relieves the competition for DAG between DGAT1 and EaDAcT to enhance acTAG accumulation. Note that the C2 fatty acid only accumulates at the sn‐3 position of the acTAG.

Figure 4

A representation of the Δ6‐pathway for biosynthesis of long‐chain polyunsaturated fatty acids in plants. The substrates linoleic acid (LA) and α‐linolenic acid (ALA) (highlighted in the box at the top) are sequentially converted through the combined activity of separate desaturase (Des) and elongase (Elo) to the target long‐chain omega‐3 fatty acids – eicosopentanoic acid (EPA) and docosahexaenoic acid (DHA). Additional enzymes with omega‐3 desaturase (ω3 Des) activities are also included to enhance the production of EPA and DHA over omega‐6 forms such as arachidonic acid (ARA) via the generic conversation of omega‐6 to omega‐3. An alternative configuration of the pathway (not illustrated) begins with a Δ9‐elongation and two rounds (Δ8, Δ5) of desaturation to generate ARA and EPA.

Figure 5

Combining synthetically engineered traits for crop improvement. The synthetic redesign of plant metabolism enables the rapid and predictable engineering of new traits in crops. Bringing these traits in a flexible chassis such as Camelina will in future enable the development of new sustainable crops for agriculture. Abbreviations: CoA, coenzyme A; Fae1, fatty acid elongase 1; G3P, glycerol‐3‐phosphate; GPAT, glycerol‐3‐phosphate acyltransferase; LPA, lysophosphatidic acid; LPAT, lysophosphatidyl acyltransferase; PA, phosphatidic acid; PAP, PAP, phosphatidic acid phosphatase; LPCAT, lysophosphatidylcholine acyltransferase; PC, phosphatidylcholine; FAD, fatty acid desaturase; TAG, triacylglycerol; DGAT, diacylglycerol acyltransferase; PDAT, phospholipid:diacylglycerol acyltransferase; PLC, phospholipase C; CPT, cholinephosphotransferase; PDCT, phosphatidylcholine:diacylglycerol cholinephosphotransferase; DAG, diacylglycerol.