scholarly journals 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

2014 ◽  
Vol 41 (1) ◽  
pp. 80 ◽  
Author(s):  
Yijun Yuan ◽  
Yinhua Chen ◽  
Shan Yan ◽  
Yuanxue Liang ◽  
Yusheng Zheng ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Nicotiana Tabacum ◽  
Acyl Carrier Protein ◽  
Cocos Nucifera ◽  
Chain Elongation ◽  
Carrier Protein ◽  
Medium Chain ◽  
Tobacco Seeds

Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acyl-acyl 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.

scholarly journals Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression

2018 ◽  
Vol 84 (10) ◽  
Author(s):  
Marika Ziesack ◽  
Nathan Rollins ◽  
Aashna Shah ◽  
Brendon Dusel ◽  
Gordon Webster ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Acyl Carrier Protein ◽  
Fatty Acyl ◽  
Carrier Protein ◽  
Fatty Acid Production ◽  
Medium Chain ◽  
Commodity Chemicals ◽  
Long Chain ◽  
Chain Fatty Acids

ABSTRACT Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 ( Cp FatB1) (C 8 specificity, lower activity) and Cp FatB2 (C 14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C 8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify Cp FatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a “positive patch” within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from Cp FatB1 into Cp FatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control. IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other detergents and could be used as biofuel precursors if production economics were favorable. Hydrocarbon-based liquid fuels must be optimized to have a desired boiling point, low freezing point, low viscosity, and other physical characteristics. Similarly, the solubility and harshness of detergents and the flexibility of plastic polymers can be modulated. The length and distribution of the carbon chains in the hydrophobic tails determine these properties. The biological synthesis of cell membranes and fatty acids produces chains of primarily 16 to 18 carbons, which give rise to current biofuels. The ultimate goal of the work presented here is to engineer metabolic pathways to produce designer molecules with the correct number of carbons in a chain, so that such molecules could be used directly as specialty commodity chemicals or as fuels after minimal processing.

scholarly journals Overexpression of the Plant Medium-chain Acyl-carrier Protein Thioesterase BTE for the Overproduction of the nC14-surfactin Isoform with Promising MEOR Applications

2021 ◽  
Author(s):  
fangxiang hu ◽  
Weijie Cai ◽  
Junzhang Lin ◽  
Weidong Wang ◽  
Shuang Li
Keyword(s):  
Fatty Acid ◽  
Hydroxy Fatty Acid ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Medium Chain ◽  
Emulsification Index ◽  
Fatty Acid Chain ◽  
Umbellularia Californica ◽  
Chain Acyl ◽  
Surfactin Production

Abstract BackgroundSurfactin, a representative biosurfactant of popeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. ResultsIn this study, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed in a recombinant surfactin production strain based on B. subtilis 168. As a result, the surfactin yield after 24 h of cultivation improved by 23%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC-MS showed that the proportion of nC14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. ConclusionThe obtained results provide an original approach to modify the fatty acid chain of surfactin and further demonstrate the importance of the length and isomerism of the β-hydroxy fatty acid chain for the MEOR application of surfactin.

scholarly journals The Two Functional Enoyl-Acyl Carrier Protein Reductases of Enterococcus faecalis Do Not Mediate Triclosan Resistance

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Lei Zhu ◽  
Hongkai Bi ◽  
Jincheng Ma ◽  
Zhe Hu ◽  
Wenbin Zhang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Enterococcus Faecalis ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Content Type ◽  
Enoyl Acp Reductase ◽  
Triclosan Resistance

ABSTRACTEnoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. TheEnterococcus faecalisgenome contains two genes annotated as enoyl-ACP reductases, a FabI-type enoyl-ACP reductase and a FabK-type enoyl-ACP reductase. We report that expression of either of the two proteins restores growth of anEscherichia colifabItemperature-sensitive mutant strain under nonpermissive conditions.In vitroassays demonstrated that both proteins support fatty acid synthesis and are active with substrates of all fatty acid chain lengths. Although expression ofE. faecalis fabKconfers toE. colihigh levels of resistance to the antimicrobial triclosan, deletion offabKfrom theE. faecalisgenome showed that FabK does not play a detectable role in the inherent triclosan resistance ofE. faecalis. Indeed, FabK seems to play only a minor role in modulating fatty acid composition. Strains carrying a deletion offabKgrow normally without fatty acid supplementation, whereasfabIdeletion mutants make only traces of fatty acids and are unsaturated fatty acid auxotrophs.IMPORTANCEThe finding that exogenous fatty acids support growth ofE. faecalisstrains defective in fatty acid synthesis indicates that inhibitors of fatty acid synthesis are ineffective in counteringE. faecalisinfections because host serum fatty acids support growth of the bacterium.

Factors Controlling Medium-Chain Fatty Acid Synthesis in Plastids from Maturing Cuphea Embryos

1993 ◽  
Vol 48 (7-8) ◽  
pp. 616-622 ◽  
Author(s):  
Jochen Fuhrmann ◽  
Klaus-Peter Heise
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Lauric Acid ◽  
Capric Acid ◽  
Acid Synthesis ◽  
Pyridine Nucleotide ◽  
Chain Elongation ◽  
Medium Chain ◽  
Chain Fatty Acids

Abstract The colorless embryos of Cuphea wrightii A. Gray accumulate capric (about 30%) and lauric acid (about 50%) in their storage lipids. Fractionation studies show that the capacities for the synthesis of these medium-chain fatty acids (MCFA) from [1-14C]acetate were strictly bound to intact plastids. These, in turn, obligately required the addition of ATP. ATP could partially be substituted by ADP. Reduction of the pyridine nucleotide pool, required for opti­mum MCFA formation within the plastids, was driven by glucose 6-phosphate. Under these conditions the plastids were capable of synthesizing MCFA like the intact tissue. The presence of CoA in the incubation medium induced acyl-CoA formation. The observed accumulation of unesterified capric and lauric acid in the absence of CoA suggests that acyl-ACP thioesterase activity is involved in the chain termination. Treatment with cerulenin led to an unexpectedly small reduction of total fatty acid synthesis while the chain elongation of capric acid was clearly inhibited. A similar accumulation of capric acid at the expense of longer chain fatty acids has been observed after replacing ATP by ADP. These findings implicate that even the condensing enzymes are involved in the control of chain ter­mination.

scholarly journals Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

2016 ◽  
Vol 84 (12) ◽  
pp. 3597-3607 ◽  
Author(s):  
Jiangwei Yao ◽  
Megan E. Ericson ◽  
Matthew W. Frank ◽  
Charles O. Rock
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Type Ii ◽  
Intracellular Growth ◽  
Content Type ◽  
Bacterial Fatty Acid

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogenListeria monocytogenesencode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype ofEscherichia colistrain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate ofL. monocytogenesin laboratory medium. Robust exogenous fatty acid incorporation was not detected inL. monocytogenesunless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth ofL. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth ofL. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth ofL. monocytogenes.

scholarly journals Lactococcus lactis fabH, Encoding β-Ketoacyl-Acyl Carrier Protein Synthase, Can Be Functionally Replaced by the Plasmodium falciparum Congener

2010 ◽  
Vol 76 (12) ◽  
pp. 3959-3966 ◽  
Author(s):  
Yu Du ◽  
Jolyn E. Gisselberg ◽  
Jacob D. Johnson ◽  
Patricia J. Lee ◽  
Sean T. Prigge ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Plasmodium Falciparum ◽  
Fatty Acid ◽  
Lactococcus Lactis ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Transcriptional Analysis ◽  
Carrier Protein ◽  
Cell Extracts ◽  
Fas Ii

ABSTRACT Plasmodium falciparum, in addition to scavenging essential fatty acids from its intra- and intercellular environments, possesses a functional complement of type II fatty acid synthase (FAS) enzymes targeted to the apicoplast organelle. Recent evidence suggests that products of the plasmodial FAS II system may be critical for the parasite's liver-to-blood cycle transition, and it has been speculated that endogenously generated fatty acids may be precursors for essential cofactors, such as lipoate, in the apicoplast. β-Ketoacyl-acyl carrier protein (ACP) synthase III (pfKASIII or FabH) is one of the key enzymes in the initiating steps of the FAS II pathway, possessing two functions in P. falciparum: the decarboxylative thio-Claisen condensation of malonyl-ACP and various acyl coenzymes A (acyl-CoAs; KAS activity) and the acetyl-CoA:ACP transacylase reaction (ACAT). Here, we report the generation and characterization of a hybrid Lactococcus lactis strain that translates pfKASIII instead of L. lactis f abH to initiate fatty acid biosynthesis. The L. lactis expression vector pMG36e was modified for the efficient overexpression of the plasmodial gene in L. lactis. Transcriptional analysis indicated high-efficiency overexpression, and biochemical KAS and ACAT assays confirm these activities in cell extracts. Phenotypically, the L. lactis strain expressing pfKASIII has a growth rate and fatty acid profiles that are comparable to those of the strain complemented with its endogenous gene, suggesting that pfKASIII can use L. lactis ACP as substrate and perform near-normal function in L. lactis cells. This strain may have potential application as a bacterial model for pfKASIII inhibitor prescreening.

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