The Other Kennedy Pathway Acyltransferases

The analysis of plant DGATs, especially the DGAT2 class, has certainly provided powerful tools for the manipulation of seed oil fatty acid content. However, the results from the FAH12/RcDGAT2 coexpression experiments suggest that even more enzymes of the Land's cycle and Kennedy pathway must be included to achieve industrially meaningful levels of novel fatty acids. The task of identifying which relevant genes to pursue is complicated by several factors:

1. Several enzymatic reactions could directly or indirectly influence the quantity and fatty acid composition of the TAG pool.

2. Only a few of the genes responsible for the enzymatic steps represented in Figure 2.1 have been cloned from any plant species; most have not been studied at the molecular level.

3. Many of the enzymes in question are present in plant genomes as large families.

The conjugases and other diverged desaturases (like tung FADX and castor hydroxylase) are typically single-copy genes, and each of the three DGAT classes seems to be represented by one or two genes. However, using the sequenced genome of Arabidopsis as a guide, many of the other lipid biosynthetic enzymes are present as large gene families. This is especially true for the glycerol-3-phosphate acyltrans-ferases (GPATs) and lysophosphatidic acid acyltransferases (LPATs). These enzymes successively catalyze the first and second glycerol acylations of the Kennedy pathway and as such are likely very important components of the channeling machinery responsible for packaging novel fatty acids into TAG. Arabidopsis contains at least eight GPAT genes [30,31] and at least five LP AT genes [32], plus several other uncharacterized genes that display homology to conserved sequence motifs common to acyltransferases of this type [33]. The protein sequences of this collection of enzymes are compared phylogenetically in Figure 2.3. The eight GPAT genes all cluster together rather tightly, while the five characterized LPATs are more highly diverged. Many of the other genes align somewhere in between these two groups,

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FIGURE 2.3 Phylogenetic comparison of known and putative GPAT (glycerol-3-phosphate acyltransferase) and LPAT (lysophosphatidic acid acyltransferase) gene families from Arabidopsis. Characterized LPAT genes are designated based on the nomenclature suggested by Kim et al. [30], while GPAT genes are named as described by Zheng et al. [28]. All other uncharacterized genes that contain acyltransferase motifs [31] are included, using their MIPS locus names. The branch lengths are proportional to the degree of divergence, with the scale of 0.1 representing 10% change.

FIGURE 2.3 Phylogenetic comparison of known and putative GPAT (glycerol-3-phosphate acyltransferase) and LPAT (lysophosphatidic acid acyltransferase) gene families from Arabidopsis. Characterized LPAT genes are designated based on the nomenclature suggested by Kim et al. [30], while GPAT genes are named as described by Zheng et al. [28]. All other uncharacterized genes that contain acyltransferase motifs [31] are included, using their MIPS locus names. The branch lengths are proportional to the degree of divergence, with the scale of 0.1 representing 10% change.

even when the tree is constructed with a true outgroup, making predictions of their possible enzymatic function almost impossible. Some of these genes (and their orthologs in other species) may indeed be useful for oilseed engineering, but much initial work must be done to define the enzymatic role, tissue-specific and temporal expression pattern, and other relevant properties of each gene before making that determination. Despite the anticipated difficulties, this task is necessary; previous studies have shown that other Kennedy pathway acyltransferases can positively affect the yield of novel fatty acids [34]. Efforts are currently under way to identify important GPAT and LPAT genes from tung tree.

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