Characterization and cloning of a stearoyl/oleoyl specific fatty acyl-acyl carrier protein thioesterase from the seeds of Madhuca longifolia (latifolia).

Deposition of oleate, stearate and palmitate at the later stages of seed development in Mahua (Madhuca longifolia (latifolia)), a tropical non-conventional oil seed plant, has been found to be the characteristic feature of the regulatory mechanism that produces the saturated fatty acid rich Mahua seed fat (commonly known as Mowrah fat). Although, the content of palmitate has been observed to be higher than that of stearate at the initial stages of seed development, it goes down when the stearate and oleate contents consistently rise till maturity. The present study was undertaken in order to identify the kind of acyl-ACP thioesterase(s) that drives the characteristic composition of signature fatty acids (oleate 37%, palmitate 25%, stearate 23%, linoleate 12.5%) in its seed oil at maturity. The relative Fat activities in the crude protein extracts of the matured seeds towards three thioester substrates (oleoyl-, stearoyl- and palmitoyl-ACP) have been found to be present in the following respective ratio 100:31:8. Upon further purification of the crude extract, the search revealed the presence of two partially purified thioesterases: a long-chain oleoyl preferring house-keeping LC-Fat and a novel stearoyl-oleoyl preferring SO-Fat. The characteristic accumulation of oleate and linoleate in the M. latifolia seed fat is believed to be primarily due to the thioesterase activity of the LC-Fat or MlFatA. On the other hand, the SO-Fat showed almost equal substrate specificity towards stearoyl- and oleoyl-ACP, when its activity towards palmitoyl-ACP compared to stearoyl-ACP was only about 12%. An RT-PCR based technique for cloning of a DNA fragment from the mRNA pool of the developing seed followed by nucleotide sequencing resulted in the identification of a FatB type of thioesterase gene (MlFatB). This gene was found to exist as a single copy in the mother plant genome. Ectopic expression of this MlFatB gene product in E. coli strain fadD88 further proved that it induced a higher level of accumulation of both stearic and oleic acids when compared to the negative control line that did not contain this MlFatB gene. It also indicated that SO-Fat indeed is the product of the MlFatB gene present in the maturing seeds of M. latifolia in nature. Additionally, a predicted 3D-structure for MlFatB protein has been developed through use of bioinformatics tools.

Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli.

A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.