Cloning, sequencing, and characterization of Escherichia coli thioesterase II

Naggert, Jürgen Κ.; Narasimhan, Meena L.; DeVeaux, Linda C.; Cho, H; Randhawa, Zafar I.; Cronan, John E.; Green, B N; Smith, Stuart

doi:10.1016/s0021-9258(18)99125-8

Cited by 84 publications

(22 citation statements)

References 30 publications

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“…Isoforms of this acyl-CoA thioesterase (ACT) 1 are present in a membrane-bound form in various subcellular organelles (Berge and Farstad, 1979;Berge et al, 1984) and in soluble form inside mitochondria (Svensson et al, 1995a), peroxisomes (Svensson et al, 1995b), and cytosol (Srere et al, 1959). The best characterized of these enzymes are those that are associated with the fatty acid-synthesizing enzyme, where ACT spec-ificity determines the chain length of the synthesized fatty acids (Naggert et al, 1991a;Tai et al, 1993;Witkowski et al, 1991). Likewise, the estrogen-induced thioesterase in duck uropygial gland has also been shown to regulate the chain lengths of fatty acid end products (Hwang and Kolattukudy, 1993).…”

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“…The mitochondrial and microsomal membrane-bound enzymes may have a similar function in controlling the chain length of the fatty acid products during the enzymatic chain elongation process (Berge, 1979). Some of these enzymes may also act as acyltransferases (Lehner and Kuksis, 1993); however, their physiological function in many systems is not known (Anderson and Erwin, 1971;Cho and Cronan, 1993;Naggert et al, 1991a). These enzymes have been purified to homogeneity from plants, animals, and bacteria, and the cDNAs encoding several of them have been cloned and sequenced (Cho and Cronan, 1993;Dormann et al, 1994;Hwang and Kolattukudy, 1993;Loader et al, 1993;Naggert et al, 1991aNaggert et al, , 1991b.…”

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confidence: 99%

“…Some of these enzymes may also act as acyltransferases (Lehner and Kuksis, 1993); however, their physiological function in many systems is not known (Anderson and Erwin, 1971;Cho and Cronan, 1993;Naggert et al, 1991a). These enzymes have been purified to homogeneity from plants, animals, and bacteria, and the cDNAs encoding several of them have been cloned and sequenced (Cho and Cronan, 1993;Dormann et al, 1994;Hwang and Kolattukudy, 1993;Loader et al, 1993;Naggert et al, 1991aNaggert et al, , 1991b. The properties, physiological regulation, and catalytic mechanisms of the purified and cloned enzymes have been extensively studied (Naggert et al, 1991a(Naggert et al, , 1991bCho and Cronan, 1993;Tai et al, 1993;Witkowski et al, 1991, Hwang andKolattukudy, 1993).…”

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Molecular Cloning and Expression of cDNA Encoding Rat Brain Cytosolic Acyl-Coenzyme A Thioester Hydrolase

Broustas

Larkins

Uhler

et al. 1996

Journal of Biological Chemistry

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The cDNA encoding rat brain cytosolic acyl-CoA thioester hydrolase (ACT) has been cloned and sequenced, and the primary structure of the enzyme has been deduced. A partial amino acid sequence (38 amino acids) of the enzyme was determined using the peptides generated after CNBr digestion of the purified enzyme. Primers synthesized on the basis of this information were used to isolate two cDNA clones, each encoding the full length of the enzyme. The nucleotide sequences of these clones contained an open reading frame encoding a 358-amino acid polypeptide with a calculated molecular mass of 39.7 kDa, similar to that determined for the purified enzyme (40.9 kDa). The deduced ACT sequence showed no homology to the known sequences of any other thioesterases nor to any other known protein sequence. However, there was a strong homology to a number of expressed sequence tag human brain cDNA clones. The identity of the ACT cDNA was confirmed by the expression of ACT activity in Escherichia coli. There was a 10-15-fold increase in ACT-specific activity in the bacterial extracts after induction with isopropyl thiogalactoside, and the properties of the expressed enzyme (fusion protein) were the same as those of the purified rat brain ACT. Northern blot analysis showed that a 1.65-kilobase ACT transcript was present in rat brain and testis but not in any other rat tissues examined. However, the ACT mRNA was induced in the liver of rats that were fed Wy-14,643, a peroxisome proliferator and inducer of rodent liver cytosolic acyl-CoA thioesterase. These results indicate that the induced rat liver ACT is homologous to the constitutive rat brain ACT.

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Molecular Cloning and Expression of cDNA Encoding Rat Brain Cytosolic Acyl-Coenzyme A Thioester Hydrolase

Broustas

Larkins

Uhler

et al. 1996

Journal of Biological Chemistry

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“…Purified Mycobacterium avium Ma TesB* has been shown to hydrolyze octanoyl-CoA and generate octanoic acid in vitro 43 . We neglected the octanoic acid synthesis activities by endogenous E. coli acyl-CoA thioesterase since Ec TesB generally has activities toward longer chain acyl-CoA (>C10) 44 and Ec YciA has activities toward shorter chain acyl-CoA (<C8) 45,46 . To test the basal octanoic acid production level, we found E. coli CM23-ΔlipB harboring pTRC99a-Pk phaG -Td TER + pACYC-Pa phaJ3 + pBTRCK-Ma tesB* plasmids produced ~20 mg/L octanoic acid after 48 hours, while the corresponding strain without Pk PhaG produced <1 mg/L octanoic acid (data not shown).…”

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confidence: 99%

Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

Yan

Cordell

Jindra

et al. 2021

Preprint

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Microbial lipid metabolism is an attractive route for producing aliphatic chemicals, commonly referred to as oleochemicals. The predominant metabolic engineering strategy centers on heterologous thioesterases capable of producing fatty acids of desired size. To convert acids to desired oleochemicals (e.g. fatty alcohols, ketones), metabolic engineers modify cells to block beta-oxidation, reactivate fatty acids as coenzyme-A thioesters, and redirect flux towards termination enzymes with broad substrate utilization ability. These genetic modifications narrow the substrate pool available for the termination enzyme but cost one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. In this work, we demonstrate an alternative acyl-transferase strategy for producing medium-chain oleochemicals. Through bioprospecting, mutagenesis, and metabolic engineering, we developed strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones using the transacylase strategy.

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“…A second Tes II, a cytosolic enzyme encoded by tesB , has broader substrate specificity and is active for C 6 to C 18 acyl-thioesters. However, since no obvious physiological or biochemical defect was observed in E. coli with tesB overexpression or deletion ( 23 , 24 ), the exact physiological function of Tes II in lipid metabolism has not been established. A third Tes III, which is encoded by fadM ( ybaW ), has been shown to be a long-chain acyl-CoA thioesterase that is most active with 3,5-tetradecadienolyl-CoA, a minor metabolite of the β-oxidation of oleic acid ( 21 ).…”

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confidence: 99%

A Futile Metabolic Cycle of Fatty Acyl Coenzyme A (Acyl-CoA) Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production

Ikeda

Takahashi

Ohtake

et al. 2021

Appl Environ Microbiol

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Fatty acyl coenzyme A (acyl-CoA) thioesterase (Tes) and acyl-CoA synthetase (FadD) catalyze opposing reactions between acyl-CoAs and free fatty acids. Within the genome of Corynebacterium glutamicum, several candidate genes for each enzyme are present, although their functions remain unknown. Modified expression of the candidate genes in the fatty acid producer WTΔfasR led to identification of one tes gene (tesA) and two fadD genes (fadD5 and fadD15), which functioned positively and negatively in fatty acid production, respectively. Genetic analysis showed that fadD5 and fadD15 are responsible for utilization of exogenous fatty acids and that tesA plays a role in supplying fatty acids for synthesis of the outer layer components mycolic acids. Enzyme assays and expression analysis revealed that tesA, fadD5, and fadD15 were coexpressed to create a cyclic route between acyl-CoAs and fatty acids. When fadD5 or fadD15 was disrupted in wild-type C. glutamicum, both disruptants excreted fatty acids during growth. Double disruption of these genes resulted in a synergistic increase in production. Additional disruption of tesA revealed a canceling effect on production. These results indicate that the FadDs normally shunt the surplus of TesA-generated fatty acids back to acyl-CoAs for lipid biosynthesis and that interception of this shunt provokes cells to overproduce fatty acids. When this strategy was applied to a high-fatty-acid producer, the resulting fadD-disrupted and tesA-amplified strain exhibited a 72% yield increase relative to its parent and produced fatty acids, which consisted mainly of oleic acid, palmitic acid, and stearic acid, on the gram scale per liter from 1% glucose. IMPORTANCE The industrial amino acid producer Corynebacterium glutamicum has evolved into a potential workhorse for fatty acid production. In this organism, we obtained evidence showing the presence of a unique mechanism of lipid homeostasis, namely, formation of a futile cycle of acyl-CoA hydrolysis and resynthesis mediated by acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD), respectively. The biological role of the coupling of Tes and FadD would be to supply free fatty acids for synthesis of the outer layer components mycolic acids and to recycle their excess to acyl-CoAs for membrane lipid synthesis. We further demonstrated that engineering of the cycle in a high-fatty-acid producer led to dramatically improved production, which provides a useful engineering strategy for fatty acid production in this industrially important microorganism.

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Cloning, sequencing, and characterization of Escherichia coli thioesterase II

Cited by 84 publications

References 30 publications

Molecular Cloning and Expression of cDNA Encoding Rat Brain Cytosolic Acyl-Coenzyme A Thioester Hydrolase

Molecular Cloning and Expression of cDNA Encoding Rat Brain Cytosolic Acyl-Coenzyme A Thioester Hydrolase

Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

A Futile Metabolic Cycle of Fatty Acyl Coenzyme A (Acyl-CoA) Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production

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