**2. Genes related to lipid metabolism in coconut palm**

Coconut palm stores oil in endosperm tissues, and its fatty acid composition changes in different developing stages of endosperm [4, 5]. The proportion of lauric acid increases with the maturing process of coconut fruit and reaches the peak when the fruit matures. The comparison of gene expression for different developing stages of endosperm indicated that the expression levels of stearoyl-acyl carrier protein desaturase, acyl-ACP thioesterase B (FatB), and lysophosphatidic acid acyltransferase (LPAAT) arose along with the endosperm development [4]. Xiao et al. [5] identified 71 genes belonging to plastidial fatty acid synthesis pathway in coconut, and 62 enzymes catalyze the conversion of pyruvate to fatty acid (**Table 1**). Moreover, the 17 plastidial proteins involved in the conversion of pyruvate to fatty acids were five- to sixfold higher in the endosperm than in the leaf or embryo tissue, such as acyl carrier protein (ACP), ketoacyl-ACP reductase (KAR), hydroxyacyl-ACP dehydratase (HAD), and pyruvate dehydrogenase complex (PDHC). TAG is a compact molecule for energy and carbon storage in organisms. Thus, another key pathway for oil storage—triglycerides (TAG) synthesis is analyzed for coconut palm and 69 genes were identified (**Table 2**). Key genes in the two pathways were deeply analyzed through in vivo and in vitro assays, including FatB, LPAAT, and orthologs of *Arabidopsis WRINKLED 1* (*WRI 1*) [9, 11, 14, 15].

#### **2.1 Genes related to MCFA accumulation in coconut endosperm**

#### *2.1.1 Acyl-acyl carrier protein thioesterases*

Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) terminate acyl chain elongation during de novo fatty acid biosynthesis. This reaction is the biochemical determinant of the fatty acid compositions of storage lipids. There are two classes of acyl-ACP TEs—FatA and FatB. Since 1996, researchers have cloned acyl-ACP TEs from California bay laurel (*Umbellularia californica*) and validated its role in accumulating MCFA by transforming it into rapeseed (*Brassica napus*). Further research has classified FatB genes into three classes based on their specificities: class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs.

**225**

*Genes Involved in Lipid Metabolism in Coconut DOI: http://dx.doi.org/10.5772/intechopen.90998*

*2.1.2 Lysophosphatidic acid acyltransferase*

accumulation of trilaurin [11].

*2.1.3 Diacylglycerol acyltransferase*

Coconut palm has two acyl-ACP thioesterase A (FatA) genes in coconut palm and five FatB genes, which were CnFatB1 (CCG011598.1), CnFatB2–1 (CCG006479.1), CnFatB2–2 (CCG007799.1), CnFatB3 (CCG019705.1), and CnFatB4 (CCG015192.1). Three FatB genes were highly expressed in more than one analyzed tissue: CnFatB2–1 (leaf and embryo), CnFatB2–2 (leaf, embryo, and endosperm), and CnFatB3 (embryo and endosperm). Three acyl-ACP TEs of coconut (CnFatB1, CnFatB2, and CnFatB3) indicated divergent specificity: CnFatB1 (JF338903) and CnFatB2 (JF338904) produced major fatty acids as myristic acid (C14:0) and palmitoleic acid (C16:1); CnFatB3 (JF338905) made mainly lauric acid (C12:0) and tetradecenoic acid (C14:1) [14]. Yuan et al. transformed and overexpressed CnFatB3 in *Arabidopsis*, and the transgenic plants increased the amounts of 12:0 (lauric acid), 14:0 (myristic acid), 16:0 (palmitic

Coconut oil has 92% saturates and most of its TAGs are trisaturated. Moreover, laurate is found enriched at sn-2 position, which is catalyzed by membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzyme. Davies et al. detected an enzyme from coconut endosperm, which is a laurate-CoA-preferring LPAAT and active during endosperm maturation [9]. The LPAAT enzyme prefers acyl-CoAs containing C10:0, C12:0, and C14:0 acyl groups as acyl-donor substrates [9]. Knutzon et al. [11] performed the LPAAT protein purification and cloned the corresponding cDNA of this gene from coconut. The gene was then transformed and expressed in *Escherichia coli*, and substrate activity profile of this gene matched that of the coconut enzyme. This copy of LPAAT is the gene named as CCG001603.1 in the first version of coconut genome sequence [5]. Knutzon et al. transformed this gene into a rapeseed transgenic gene line which is expressed of a California bay laurel (*Umbellularia californica*) 12:0-acyl carrier protein thioesterase (BET) and contained up to 50% laurate in its developing seeds [11]. In this transgenic rapeseed with BTE, laurate is found almost exclusively at the *sn-1* and *sn-3* positions of the triacylglycerols. Coexpression of the coconut *LPAAT* gene in the transgenic rapeseeds facilitates efficient laurate deposition at the *sn-2* position and caused the

Xu et al. cloned the promoter sequence of the LPAAT gene and characterized the promoter by constructing a series of plasmids with promoter sequences with varied length of deletions to promote a β-glucuronidase (GUS) gene. The plasmids were transformed into rice, and the transgenic plants showed that reporter genes with these promoter fragments tend to express specifically in rice endosperm [12]. Yuan et al. transformed *CnLPAAT* into yeast, and tested fatty acid composition indicated that the gene increased the levels of C12:0 and C14:0 in a CnLPAAT-pYES2 transformant [16]. However, heterologous overexpression of CnLPAAT in tobacco (*Nicotiana tabacum* L.) decreased the contents of C12:0 and C14:0 in transgenic tobacco seeds, which could result from low contents of short- and medium-chain

FAs (0.22%), which are available in tobacco seeds of the total FAs.

Besides genes important for MCFA accumulation, there are key genes in TAG biosynthesis pathway that influence oil contents and FA composition.

Diacylglycerol acyltransferases (DGAT) and phospholipid:diacylglycerol acyltransferases (PDAT) catalyze diacylglycerol (DAG) to form TAG as the final step in TAG synthesis, using either acyl-CoAs or phospholipids. DAG is an important branch

acid), and 18:0 (stearic acid) by 30-, 80-, 4-, and 2-fold, respectively [6].

*Genes Involved in Lipid Metabolism in Coconut DOI: http://dx.doi.org/10.5772/intechopen.90998*

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

for food and other applications. The most noticeable feature of coconut oil is that the major components of fatty acids are medium-chain fatty acid. This feature has attracted the attention of researchers and become the focus of coconut oil research. What genes related with the accumulation of medium-chain fatty acid in endosperm? How these genes were evolved and related to a closely related species—oil palm (*Elaeis guineensis*), which also has MCFA as its main fatty acid component in

We had reviewed three parts of research related to coconut lipid metabolism in this chapter. Firstly, we summarized key genes related to MCFA accumulation in coconut endosperm. Secondly, we summarized the evolutionary relationship between coconut palm and oil palm for MCFA accumulation. Thirdly, we include descriptions of in vivo and in vitro gene validation experiments. Two tables provide coconut genes related to de novo fatty acid biosynthesis (**Table 1**) and triacylglycer-

Coconut palm stores oil in endosperm tissues, and its fatty acid composition changes in different developing stages of endosperm [4, 5]. The proportion of lauric acid increases with the maturing process of coconut fruit and reaches the peak when the fruit matures. The comparison of gene expression for different developing stages of endosperm indicated that the expression levels of stearoyl-acyl carrier protein desaturase, acyl-ACP thioesterase B (FatB), and lysophosphatidic acid acyltransferase (LPAAT) arose along with the endosperm development [4]. Xiao et al. [5] identified 71 genes belonging to plastidial fatty acid synthesis pathway in coconut, and 62 enzymes catalyze the conversion of pyruvate to fatty acid (**Table 1**). Moreover, the 17 plastidial proteins involved in the conversion of pyruvate to fatty acids were five- to sixfold higher in the endosperm than in the leaf or embryo tissue, such as acyl carrier protein (ACP), ketoacyl-ACP reductase (KAR), hydroxyacyl-ACP dehydratase (HAD), and pyruvate dehydrogenase complex (PDHC). TAG is a compact molecule for energy and carbon storage in organisms. Thus, another key pathway for oil storage—triglycerides (TAG) synthesis is analyzed for coconut palm and 69 genes were identified (**Table 2**). Key genes in the two pathways were deeply analyzed through in vivo and in vitro assays, including FatB, LPAAT, and orthologs of

**224**

endosperm?

ols (TAG) biosynthesis (**Table 2**).

**2. Genes related to lipid metabolism in coconut palm**

*Arabidopsis WRINKLED 1* (*WRI 1*) [9, 11, 14, 15].

*2.1.1 Acyl-acyl carrier protein thioesterases*

**2.1 Genes related to MCFA accumulation in coconut endosperm**

Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) terminate acyl chain elongation during de novo fatty acid biosynthesis. This reaction is the biochemical determinant of the fatty acid compositions of storage lipids. There are two classes of acyl-ACP TEs—FatA and FatB. Since 1996, researchers have cloned acyl-ACP TEs from California bay laurel (*Umbellularia californica*) and validated its role in accumulating MCFA by transforming it into rapeseed (*Brassica napus*). Further research has classified FatB genes into three classes based on their specificities: class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs.

Coconut palm has two acyl-ACP thioesterase A (FatA) genes in coconut palm and five FatB genes, which were CnFatB1 (CCG011598.1), CnFatB2–1 (CCG006479.1), CnFatB2–2 (CCG007799.1), CnFatB3 (CCG019705.1), and CnFatB4 (CCG015192.1). Three FatB genes were highly expressed in more than one analyzed tissue: CnFatB2–1 (leaf and embryo), CnFatB2–2 (leaf, embryo, and endosperm), and CnFatB3 (embryo and endosperm). Three acyl-ACP TEs of coconut (CnFatB1, CnFatB2, and CnFatB3) indicated divergent specificity: CnFatB1 (JF338903) and CnFatB2 (JF338904) produced major fatty acids as myristic acid (C14:0) and palmitoleic acid (C16:1); CnFatB3 (JF338905) made mainly lauric acid (C12:0) and tetradecenoic acid (C14:1) [14]. Yuan et al. transformed and overexpressed CnFatB3 in *Arabidopsis*, and the transgenic plants increased the amounts of 12:0 (lauric acid), 14:0 (myristic acid), 16:0 (palmitic acid), and 18:0 (stearic acid) by 30-, 80-, 4-, and 2-fold, respectively [6].
