Production of Novel Oils in Plants Denis J Murphy
Total Page：16
File Type：pdf, Size：1020Kb
175 Production of novel oils in plants Denis J Murphy We have now isolated the great majority of genes encoding unexpected ways. We have seen the isolation of some enzymes of storage oil biosynthesis in plants. In the past two potentially key genes that contribute both to the quantity years, particular progress has been made with acyltransferases, and quality of seed storage oils. There has also been an ketoacyl-acyl carrier protein synthetases and with desaturases increasing appreciation of the importance of fatty acids, and their relatives. In some cases, these enzymes have been re- not only as storage or structural components, but also act- engineered to create novel products. Nevertheless, the single or ing as, or giving rise to, important signalling molecules that multiple insertion of such transgenes into oil crops has not always regulate many aspects of plant development [1,2]. This led to the desired phenotype. We are only now beginning to illustrates the importance of ensuring that novel fatty acids appreciate some of the complexities of storage and membrane in transgenic oil crops are correctly targeted to the storage lipid formation, such as acyl group remodelling and the turnover oil and are hence unable to adversely affect membrane or of unusual fatty acids. This understanding will be vital for future signalling functions. The purpose of this article is to attempts at the rational engineering of transgenic oil crops. In review some of the recent progress in understanding the parallel with this, the domestication of plants already synthesising mechanism and regulation of storage oil formation in useful fatty acids should be considered as a real alternative to the plants, and how this may impact on its biotechnological transgenic approach to producing novel oil crops. manipulation. Addresses Industrial and edible oils Brassica and Oilseeds Research Department, John Innes Centre, Before reviewing some of the recent technical develop- Norwich Research Park, Norwich NR4 7UH, UK; ments, it may be useful to consider what we are trying to e-mail: [email protected] achieve in modifying seed oils and why we are doing it. At Current Opinion in Biotechnology 1999, 10:175–180 present, over 80% of the 75 million tonnes of globally trad- ed seed oils are used for edible purposes, most notably in http://biomednet.com/elecref/0958166901000175 the production of cooking oils, margarines and processed © Elsevier Science Ltd ISSN 0958-1669 foods [3]. Global production of plant oils for industrial use Abbreviations (i.e. oleochemicals) is only about 15 million tonnes per year ACP acyl carrier protein with a value of about $400–800 per tonne. This contrasts DGAT diacylglycerol acyltransferase with the pre-tax price of refined petroleum products which DHA docosahexenoic acid are produced in the hundreds of millions of tonnes at a EPA eicosapentenoic acid price of only $100–300 per tonne. These simple economic TAG triacylglycerol factors mean that oleochemicals from transgenic oil crops can only realistically succeed at present either as high- Introduction value niche products for very specific applications (e.g. The manipulation of seed oil content via transgene inser- 8-linolenic acid as a therapeutic agent) or by competing on tion was one of the early successful applications of modern a larger scale with petrochemicals by virtue of higher puri- biotechnology in agriculture. Indeed, the first transgenic ty, better performance and/or environmental benefits. Of crop with a modified seed composition to be approved for course, oleochemicals will eventually become more com- unrestrictive commercial cultivation in the USA was a lau- petitive with petrochemicals as global reserves of ric oil rapeseed grown in 1995. There are two major reasons fossil-derived hydrocarbons (oil, coal and gas) begin to run for this. Firstly, rapeseed, Brassica napus, is a species that is out in the coming century [4•]. relatively amenable to transformation and regeneration, whereas many other major crops have proved more recalci- The manipulation of oils for new types of edible, rather than trant. Secondly, the metabolic pathways involved in industrial, use appears much more restricted as the vast storage oil biosynthesis appeared at first to be well defined majority of plant lipids are already regarded as desirable and potentially straightforward to manipulate via single dietary components, with better nutritional qualities than gene insertions. animal fats. There are, however, two obvious targets for mod- ified edible oils in seeds. Firstly, to manipulate the ratio of Nevertheless, much of the early optimism for producing saturated to polyunsaturated fatty acids in order to avoid the designer oilseeds has, over recent years, been tempered by need for chemical hydrogenation, which produces the high setbacks in obtaining high yields of specific novel fatty levels of trans-fatty acids that many believe to be undesirable acids in transgenic oilseed crops. During the past two in the diet [5]. Secondly, there is increasing interest in pro- years, there has been an increasing recognition of the com- ducing very long-chain polyunsaturates, such as plexity of the metabolic pathways involved in seed oil docosahexenoic acid (DHA) and eicosapentenoic acid (EPA), biosynthesis and several new enzymes have been discov- which are nutritionally beneficial as precursors for certain ered that contribute to these processes in quite prostaglandins and as cholesterol-lowering agents [6]. These 176 Plant biotechnology fatty acids are particularly enriched in fish oils but are now in genes encoding the vast majority of these enzymes. Some increasingly short supply due to the depletion of the world’s of the most significant developments have taken place in fish stocks. A recent advance here is the isolation of a gene characterising desaturases and related diiron-oxo proteins ∆ • encoding a 5 desaturase from the yeast Mortierella aplina [7]. in plants, as recently reviewed in detail [13 ]. This enzyme is responsible for the conversion of di-homo-γ- linolenic acid to arachidonic acid which is the central It now appears that plants contain two major families of precursor for the production of eicosonoids, such as diiron-oxo enzymes. Firstly, there are the soluble plastid- prostaglandins, leukotrienes and thromboxanes. localised acyl-acyl carrier protein (ACP) desaturases that typically work on C14, C16 and C18 saturated acyl-ACP While the manipulation of oil crops for human consumption substrates. These plant desaturases have similar tertiary has many attractions, this is an acutely sensitive topic of structures and ligand-binding sites to microbial methane public concern at present, particularly in the case of new monooxygenases and ribonucleotide reductases and fall food products. For example, petroselinic acid, which can into class II of the diiron-oxo enzymes [14]. One of these serve as a useful industrial raw material for polymer and plant enzymes, ∆-9 stearoyl-ACP desaturase, was the first detergent manufacture, has also been proposed as a harden- desaturase from any organism for which a high resolution ing agent for margarines [8]. Dietary studies in rats, crystal structure (down to 2.4 Å) was obtained [15]. This however, indicate that petroselinic acid ingestion is associat- knowledge has considerably assisted efforts to engineer ed with liver abnormalities and inhibition of arachidonic novel positional and chain-length specificities into desat- acid biosynthesis [9,10]. This case illustrates the unforeseen urases, for example, via site-directed mutagenesis. For difficulties that may arise from the introduction of novel example, using information from the crystal structure, a (particularly transgenic-derived) oils into the diet and indi- ∆-9 stearoyl-ACP desaturase was converted into an cates that such transgenic oil crops may be better targeted enzyme with a substrate preference for palmitoyl-ACP by initially to produce industrial, rather than edible, products. the replacement of two residues (Leu118→Phe and Pro179→Ile) [16••]. In a parallel study, the single site- Non-oil products directed mutagenesis of residue Leu118→Trp resulted in In addition to producing seed oils with novel fatty acid com- the conversion of a stearoyl-ACP desaturase to an enzyme positions, there are numerous other actual or potential with an 80-fold increase in specificity for palmitoyl-ACP applications of transgenic oil crops. For example, as recent- [17]. This represents one of the first successful attempts at ly reviewed [11], following the insertion of a relatively small the rational modification of an enzyme of lipid biosynthe- number of genes from certain bacteria, such as Alcaligenes sis. In the future, this approach holds great promise for the spp, carbon can be diverted from oil synthesis towards the re-engineering of desaturases and other enzymes for the accumulation of polyhydroxyalkanoates. These polyesters production of novel fatty acids in transgenic oil crops. are biodegradable thermoplastics. Their use is currently lim- ited by their high price (up to tenfold higher than The second type of plant desaturase falls into class III of conventional plastics) due to the high cost of their manufac- the diiron-oxo enzymes; all such proteins are membrane- ture via bacterial fermentation. Significant reductions in the bound and utilise either complex lipid or acyl-CoA price of such biodegradable polyesters could be expected if substrates. It has recently been demonstrated that several