Cloning of a palmitoyl-acyl carrier protein thioesterase from oil palm

Abstract

A palmitoyl-acyl carrier protein (ACP) thioesterase cDNA clone was isolated from an oil palm cDNA library. The cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and a crude bacterial extract was assayed for acyl-CoA-hydrolysing activity. The recombinant enzyme was able to hydrolyse medium- and long-chain acyl-CoAs. Northern-blot analysis showed a high level of gene expression in leaf, flower and 15-, 17- and 18-week mesocarp tissues. Low-level gene expression was detected in germinated seedlings and 8- and 12-week mesocarp tissues, but no transcript was detected in any kernel tissues. Southern-blot analysis indicated the presence of a single gene and we have also isolated a genomic clone using the cDNA as a probe. Two genomic fragments were subcloned and a 7 kb contiguous stretch of the oil palm genome was sequenced. Comparison of this sequence with the cDNA sequence identified a putative 93 amino acid transit peptide, most of which is missing from the cDNA. The coding region of the gene consisted of seven exons and six introns.

Molecular cloning and characterisation of an acyl carrier protein thioesterase gene (CocoFatB1) expressed in the endosperm of coconut (Cocos nucifera) and its heterologous expression in Nicotiana tabacum to engineer the accumulation of different fatty acids

“…Compared with the results of function analysis of CnFatB1, CocoFatB1 is specific not only towards 14 : 0-ACP and 16 : 0-ACP, which have been demonstrated in E. coli by Jing et al (2011), but also showed specificity to 18 : 0-ACP in plant. These results are similar to those previously reported for FatB thioesterases from other plants, such as Elaeis guineensis (Othman et al 2000), Jatropha curcas L. (Wu et al 2009), Cuphea hookeriana (Jones et al 1995), Diploknema (Madhuca) butyracea (Jha et al 2006) and Indian mustard (Brassica juncea L. Czern.…”

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

“…Secreted fatty acids were analyzed with GC-MS, and the total fatty acid yield in the medium was used to represent the in vivo activities of these enzymes on acyl-ACPs, though it remains possible that some of these enzymes might also hydrolyze acyl-CoAs [43]. …”

Comparative Transcriptome Analysis of Three Oil Palm Fruit and Seed Tissues That Differ in Oil Content and Fatty Acid Composition

“…Arabidopsis FatB displayed the highest activity toward 16:0-ACP but also displayed significant activity with 18:1-ACP (about 75% of that obtained with 16:0-ACP; Salas and Ohlrogge, 2002). Substrate specificity of only one oil palm FatB enzyme was characterized until now (Othman et al, 2000). Its sequence corresponded to the paralog EgFatB2 from this work and the recombinant enzyme obtained from this clone displayed a marked preference for 16:0 over 12:0 and 18:1.…”