**4.2 Testing enzyme activity in vivo**

Gene function validation has been conducted through gene overexpression in heterologous plant systems which have stable gene transformation system, such as *Arabidopsis*, rice, and tobacco. Functional characterization of *CnWRI1* was done by gene overexpression in *Arabidopsis* and rice [15]. Overexpression of CnWRI1 in *Arabidopsis* seeds caused fatty acid composition changes but not for oil content, while overexpression of the gene in rice endosperm increased the starch content and decreased the protein contents [15]. For gene function validation of CnLPAAT (CCG001603.1), this gene was overexpressed in a transgenic oilseed (*Brassica napus*) plant, which expressed a 12:0-ACP thioesterase from California bay laurel (*Umbellularia californica*). The transgenic lines that coexpressed a 12:0-ACP thioesterase and CnLPAAT had increase laurate content from 50 mol% to total laurate levels, which suggested that CnLPAAT facilitates efficient laurate deposition at the sn-2 position [11].

Transient transgenic expression system of tobacco is also widely used for gene function analysis. Genes belonging to lipid metabolism were also validated by this system, investigating the possibility of oil production in non-sees biomass [18].

*Escherichia coli* (*E. coli*) strains are commonly used in molecular biology, because the introduction of DNA into *E. coli* is convenient. Since lipid metabolism is basic in all living cells, specific *E. coli* strain with gene mutation could be used for analyzing enzyme functions. Knutzon et al. [11] cloned the CnLAAPT gene copy (CCG001603.1) from coconut endosperm and tested enzyme activity by introducing the gene into *E. coli* strain K27 that has a mutation in the *fadD* gene as well as β-oxidation of fatty acids. Overexpression of this *CnLAAPT* gene copy caused the accumulation of free fatty acids in the growth medium. Enzymic specificity of three acyl-ACP TEs of coconut (CnFatB1, CnFatB2, and CnFatB3) have been tested by transforming and expressing in *E. coli* K27 and analyzing free fatty acids accumulated in the medium [14].
