Metabolic Engineering of Lipids: A Close View

This article provides a close view on metabolic engineering of lipids.

Plant oils are useful as foods (about two thirds), and for industrial purposes (about one third). The industrial applications of plant oils include their use in the manufacture of soaps, detergents, lubricants and biofuels.

An outline of the synthesis of oils (chemically triacylglycerol’s) is depicted in Fig 51.2. There is an involvement of plastids, cytoplasm and endoplasmic reticulum for the production of oils. After their synthesis, oils are stored in lipid bodies.

Acetate from the cytoplasm is taken up by the plastids and converted to acetyl CoA and malonyl CoA. These two molecules undergo a series of reactions, catalysed by the enzyme fatty acid synthase (FAS), to produce fatty acyl carrier proteins with different carbons atoms.

Some examples are listed:

i. Laurate — C₁₂

ii. Myristate — C₁₄

iii. Palmitate — C₁₆

iv. Stearate — C₁₈

v. Oleate — C_18:1

By the action of acyl-ACP thioesterases, the fatty acids are released and exported to the cytoplasm. Here, they may undergo certain modifications— elongation, desaturation (insertion of double bonds), hydroxylation etc.

These modified fatty acids react with glycerol 3-phosphate (in the endoplasmic reticulum) to finally form triacylglycerol’s (TG). TG are transported to lipid oil bodies and stored. The oil bodies of seeds also contain proteins called oleosins, in the lipid monolayers. Some of the approaches of genetic engineering for the manipulation of plant oil biosynthesis are briefly described.

Production of Shorter Chain Fatty Acids:

Majority of the plant oils contain more than 16-carbons e.g. palmitic, stearic, oleic acids. Oils with shorter chains (C₈—C₁₄) are more useful in many industries — for the production of soaps, detergents, cosmetics etc.

It is possible to terminate the hydrolysis of acyl- ACP by specific thioesterases and produce high proportion of selected fatty acids. One acyl-ACP thioesterase that specifically hydrolyses lauroyl-ACP has been isolated from California bay tree (Umbellularia californica). The gene encoding this enzyme has been cloned and transferred to oilseed rape. The transgenic plants were found to produce oils with high proportion of lauric acid (12-carbon fatty acid).

Production of Longer Chain Fatty Acids:

For the application as industrial oils, triacylglycerol’s with longer chain fatty acids (>C₁₈) are preferred. Thus, erucic acid (22C) containing oils are useful in industries. However, erucic acid is unsuitable for human consumption. By conventional plant breeding two distinct crops of oilseed rape have been developed — high-erucic acid rape (HEAR) and low-erucic acid rape (LEAR).

Some attempts are being made to genetically manipulate crops to yield oils with high contents of erucic acid. The approaches include to overexpress genes encoding the enzymes elongases and transfer of genes to produce enzymes that can preferentially incorporate erucic acid into triacylglycerol’s.

Production of Fatty Acids with Modified Degree of Saturation:

A fatty acid is said to be saturated if it has no double bonds. Saturated and unsaturated (with on or more double bonds) fatty acids occur in nature. It is possible to genetically manipulate the degree of saturation in fatty acids.

Production of unsaturated fatty acids:

Oleic acid (C₁₈: 1) rich oils are useful as food, feed and in some industries. There has been some success in the transfer of antisense gene encoding the enzyme desaturase in oilseed rape and soybean plants. These transgenic plants were found to produce oils with very high proportion of oleic acid.

Production of saturated fatty acids:

Sometimes, it is desirable to produce oils with high contents of saturated fatty acids. Success has been reported by use of antisense RNA approach. The objective was to reduce the conversion of stearate (C₁₈: 0) to unsaturated fatty acids such as oleic acid (C₁₈: 1), linoleic acid (C₁₈₁₈: 2) and linolenic acid (C₁₈: 3).

The enzyme stearoyl-ACP desaturase catalyses the conversion of stearoyl-ACP to oleoyl-ACP. The gene encoding the enzyme stearoyl-ACP desaturase has been isolated from Brassica rapa and its complementary sequence cloned. The transgenic plants developed by this approach were found to contain high contents of saturated fatty acid namely stearic acid, and low concentration of oleic acid.

Production of Rare Fatty Acids:

There are some rare fatty acids, which are industrially important, but not normally synthesized in the plants. Some plants producing the rare fatty acids have been identified and the genes transferred for their overproduction.

One good example is the production of petro-selenic acid that is found in coriander. Tobacco plants were transformed with coriander acyl-ACP desaturase that led to a high production of petro-selenic acid in tobacco plants. Petro-selenic acid is commercially important. On oxidation by ozone, it forms lauric acid (for use in soap and detergent production) and adipic acid (for the manufacture of nylon).

Some success has also been reported in the increased production of several unsaturated fatty acids through genetic manipulation by incorporating genes coding special enzymes namely front-end desaturases e.g. γ-linolenic acid, arachidonic acid, ricolenic acid.

Biodegradable Plastics (Bio-Plastics):

Biodegradable plastics (or bio-plastics) are chemically polyhydroxy alkanoates (PHAs). They are currently being produced in large quantities by microbial fermentation. Among the PHAs, polyhydroxy butyrate (PHB) is the most important one. Several experimental studies are in progress to produce bulk quantities of bio-plastics in plants. Two approaches are described hereunder (Fig 51.3).

PHB production in cytoplasm:

Starting from acetyl CoA, polyhydroxybutyrate is a three-stage pathway, involving the following enzymes (with corresponding gene).

i. 3-Ketothiolase (phaA).

ii. Acetoacetyl CoA reductase (phaB).

iii. PHB synthase (phaC).

The three genes coding the respective enzymes have been isolated from Alcaligenes eutrophus and cloned. The cytoplasm of plant cell (e.g. Arabidopsis) contains 3-ketothiolase. Therefore, in the initial experiments only two genes (phaB and phaC) coding acetoacetyl CoA reductase and PHB synthase were transferred to develop Arabidopsis (Fig 51.3A). By this approach, the quantity of BHP produced was very low. Another limitation was that the plants had stunted growth.

PHB production in plastids:

The problems encountered in the above approach were overcome by transferring all three genes (phaA, phaB, phaQ of PHB synthesis and targeting them to chloroplast (Fig 51.3B). To achieve this, each gene was separately fused with a coding sequence of transit peptid bound to N-terminal fragment of Rubisco (ribulose 1,5- bisphosphate carboxylase) subunit protein. The genes expression was carried out by CaMV 35S promoter.

Transgenic Arabidopsis plants with each gene construct were first developed. Then a series of sexual crossings were carried out between the individual trans-formants. The transgenic plants developed by this approach yielded good quantity of bio-plastics (about 14% dry weight of the plant), and in addition there was no observable adverse effect on the growth or fertility of these plants.

Production of bio-plastics in cotton fibres :

Cotton fibres contains the enzyme β-ketothiolase. Therefore, the genes for the other two enzymes of PHB pathway (phaB and phaQ from Alcaligenes eutrophus were transferred into meristems of cotton plant by particle bombardment. Adequate synthesis of PHB was reported in the fibres of transgenic cotton plants.

Production of polyhydroxyalkanoate co-polymers:

The most important bio-plastic of commercial importance is polyhydroxybutyrate. The genetic engineering approaches for its manufacture in plants are described above. But the disadvantage with PHB production is that it is found as stiff and bristle plastics, since it forms highly crystalline polymers.

The other bio-plastic, composed of polylydroxyalkanoate (PHA) co-polymer is a polymer made up of longer monomers. It is less crystalline and more flexible compared to PHB. The PHAs are produced from the intermediates of β-oxidation of fatty acids-notably β-hydroxy acyl CoA. Some success has been reported in the production of PHAs through genetic manipulations of peroxisomes and glyoxisomes.