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

Jha, J.K.; Maiti, Mrinal K.; Bhattacharjee, A.; Basu, Ananjan; Sen, Parimal C.; Sen, Sandeep

doi:10.1016/j.plaphy.2006.09.017

Cited by 36 publications

(41 citation statements)

References 32 publications

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“…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.…”

Section: Discussionsupporting

confidence: 91%

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

Yang

Chen

Liang

et al. 2014

Functional Plant Biol.

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Abstract. Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acylacyl carrier protein (ACP) thioesterases that hydrolyse acyl-ACP into free fatty acids to terminate acyl chain elongation during fatty acid biosynthesis. A full-length cDNA of an acyl-ACP thioesterase, designated CocoFatB1, was isolated from cDNA libraries prepared from coconut endosperm during fruit development. The gene contained an open reading frame of 1254 bp, encoding a 417-amino acid protein. The amino acid sequence of the CocoFatB1 protein showed 100% and 95% sequence similarity to CnFatB1 and oil palm (Elaeis guineensis Jacq.) acyl-ACP thioesterases, respectively. Real-time fluorescent quantitative PCR analysis indicated that the CocoFatB1 transcript was most abundant in the endosperm from 8-month-old coconuts; the leaves and endosperm from 15-month-old coconuts had~80% and~10% of this level. The CocoFatB1 coding region was overexpressed in tobacco (Nicotiana tabacum L.) under the control of the seed-specific napin promoter following Agrobacterium tumefaciens-mediated transformation. CocoFatB1 transcript expression varied 20-fold between different transgenic plants, with 21 plants exhibiting detectable levels of CocoFatB1 expression. Analysis of the fatty acid composition of transgenic tobacco seeds showed that the levels of myristic acid (14 : 0), palmitic acid (16 : 0) and stearic acid (18 : 0) were increased by 25%, 34% and 17%, respectively, compared with untransformed plants. These results indicated that CocoFatB1 acts specifically on 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP, and can increase medium chain saturated fatty acids. The gene may valuable for engineering fatty acid metabolism in crop improvement programmes.

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Section: Discussionsupporting

confidence: 91%

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

Yang

Chen

Liang

et al. 2014

Functional Plant Biol.

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“…It should be noted that plant thioesterases have long been characterized by their expression in ␤-oxidation-deficient E. coli strains (20,35,37,39,41). The acids released to the culture medium (both unsaturated and saturated acids of chain lengths 8 -18 carbon atoms) accurately reflect the substrate specificities observed for the purified enzymes in vitro (20,35,37,39,(41)(42)(43)(44). When examined export was essentially quantitative, although no discrete export system has been identified.…”

Section: Journal Of Biological Chemistry 29531mentioning

confidence: 99%

Escherichia coli Unsaturated Fatty Acid Synthesis

Feng

Cronan

2009

Journal of Biological Chemistry

166

108

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Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the years. The key players are the fabA and fabB genes. Earlier studies of fabA transcription showed that the gene was transcribed from two promoters, with one being positively regulated by the FadR protein. The other weaker promoter (which could not be mapped with the technology then available) was considered constitutive because its function was independent of FadR. However, the FabR negative regulator was recently shown to represses fabA transcription. We report that the weak promoter overlaps the FadR-dependent promoter and is regulated by FabR. This promoter is strictly conserved in all E. coli and Salmonella enterica genomes sequenced to date and is thought to provide insurance against inappropriate regulation of fabA transcription by exogenous saturated fatty acids. Also, the fabAup promoter, a mutant promoter previously isolated by selection for increased FabA activity, was shown to be a promoter created de novo by a four-base deletion within the gene located immediately upstream of fabA. Demonstration of the key UFA synthetic reaction catalyzed by FabB has been elusive, although it was known to catalyze an elongation reaction. Strains lacking FabB are UFA auxotrophs indicating that the enzyme catalyzes an essential step in UFA synthesis. Using thioesterases specific for hydrolysis of short chain acyl-ACPs, the intermediates of the UFA synthetic pathway have been followed in vivo for the first time. These experiments showed that a fabB mutant strain accumulated less cis-5-dodecenoic acid than the parental wild-type strain. These data indicate that the key reaction in UFA synthesis catalyzed by FabB is elongation of the cis-3-decenoyl-ACP produced by FabA.The fabA gene of Escherichia coli encodes 3-hydroxydecanoyl-acyl carrier protein (ACP) 2 dehydratase/isomerase, a bifunctional enzyme that introduces the unsaturated fatty acid (UFA) double bond at the C10 level (1-3) (see Fig. 1). As expected fabA mutants require supplementation with UFA for growth (4). Although UFA synthesis can be considered a housekeeping function, fabA transcription is subject to an unexpectedly complex regulation. Work from this laboratory and others showed that fabA transcription is positively regulated by FadR, a protein that also functions as the repressor of the ␤-oxidation regulon (5-7). In that work the FadR-dependent promoter was identified as well as a second weaker promoter that was unaffected by FadR and thus was considered constitutively active (5, 6). The FadR-independent promoter is thought to ensure continued expression of fabA under conditions where FadR activation is compromised. E. coli fabA mutants lyse in the absence of UFA (3). Because FadR responds to CoA thioesters of both unsaturated and saturated fatty acids, E. coli could shut down UFA synthesis in response to saturated fatty acid (SFA) availability, a potential disaster. We have proposed that E. ...

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“…Unfortunately factors that in turn control the release of free FA from ACP are poorly understood. Alternatively one could isolate the heterologous FAT genes from other oil seed plants (Dormann et al 1995;Hawkins and Kridl 1998;Jha et al 2006) and overexpress in sesame.…”

Section: Fatty Acid Desaturase Gene(s) In Plants and Their Functionmentioning

confidence: 99%

Metabolic engineering of fatty acid biosynthetic pathway in sesame (Sesamum indicum L.): assembling tools to develop nutritionally desirable sesame seed oil

2015

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Vegetable oils are an essential component of human diet, in terms of their health beneficial roles. Despite their importance, the fatty acid profile of most commonly used edible oil seed crop plants are imbalanced; this skewed ratio of fatty acids in the diet has been shown to be a major reason for the occurrence of cardiovascular and autoimmune diseases. Until recently, it was not possible to exert significant control over the fatty acid composition of vegetable oils derived from different plants. However, the advent of metabolic engineering, knowledge of the genetic networks and regulatory hierarchies in plants have offered novel opportunities to tailor-made the composition of vegetable oils for their optimization in regard to food functionality and dietary requirements. Sesame (Sesamum indicum L.) is one of the ancient oilseed crop in Indian subcontinent but its seed oil is devoid of balanced proportion of x-6:x-3 fatty acids. A recent study by our group has shed new lights on metabolic engineering strategies for the purpose of nutritional improvement of sesame seed oil to divert the carbon flux from the production of linoleic acid (C18:2) to a-linolenic acid (C18:3). Apart from that, this review evaluates current understanding of regulation of fatty acid biosynthetic pathways in sesame and attempts to identify the major options of metabolic engineering to produce superior sesame seed oil.Keywords 1,2-sn-diacylglycerol acyltransferase Á Fatty acid desaturase Á Fatty acyl-ACP thioesterase Á Sesame seed oil Á Stearoyl-acyl-carrier protein D9-desaturase Abbreviations ACCase Acetyl Co-A carboxylase ADP Adenosine diphosphate ATP Adenosine triphosphate CVD Cardiovascular diseases DAG 1,2-diacylglycerol DGAT 1,2-sn-diacylglycerol acyltransferase ER Endoplasmic reticulum FA Fatty acid FAD3 Fatty acid desaturase-3 FAD7 Fatty acid desaturase-7 FAE Fatty acid elongase FAS Fatty acid synthase

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

Cited by 36 publications

References 32 publications

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

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

Escherichia coli Unsaturated Fatty Acid Synthesis

Metabolic engineering of fatty acid biosynthetic pathway in sesame (Sesamum indicum L.): assembling tools to develop nutritionally desirable sesame seed oil

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