Thioesterase II of
            <i>Escherichia coli</i>
            Plays an Important Role in 3-Hydroxydecanoic Acid Production

Zheng, Zhong; Gong, Qiang; Liu, Tao; Deng, Ying; Chen, Jinchun; Chen, Guoqiang

doi:10.1128/aem.70.7.3807-3813.2004

Cited by 48 publications

(34 citation statements)

References 34 publications

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“…To find thioesterases that increase specificity for and production of 3-hydroxy, unsaturated, saturated, and branched fatty acids in the pathway, 62 putative thioesterases were chosen for screening. A group of six thioesterases, three TesB and three TesB2 enzymes, were chosen that had 38 to 50% amino acid identity to E. coli MG1655 TesB (EcTesB), which makes 3-hydroxy and saturated fatty acids in vivo (1,6,29), and 40 to 41% amino acid identity to A. borkumensis TesB2 enzyme, which was previously described as having specificity for 3-hydroxy acyl-CoAs (30) ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

McMahon

Prather

2014

Appl Environ Microbiol

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b Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we conducted in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate), and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. In this study, we introduce novel thioesterases with improved specificity for saturated, branched, and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent.

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Section: Resultsmentioning

confidence: 99%

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

McMahon

Prather

2014

Appl Environ Microbiol

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“…Furthermore, for most of the known functions, they participate in metabolic pathways, such as phenolic compound degradation or fatty acid or siderophore synthesis pathways. For example, PaaI and MaoC are involved in phenylacetic acid degradation (9), TesB may be used to enhance 3-hydroxydecanoic acid production from polyhydroxyalkanoates in E. coli (42), and a tesB-like gene has also been involved in polyhydroxyalkanoate metabolism in Alcanivorax borkumensis (35). These enzymatic activities are of great interest for biotechnological applications, which is certainly why the unknown-function proteins of the hotdog family have been all targeted in structural genomic projects.…”

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confidence: 99%

The Hotdog Thioesterase EntH (YbdB) Plays a Role In Vivo in Optimal Enterobactin Biosynthesis by Interacting with the ArCP Domain of EntB

2007

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In response to iron limitation, the siderophore enterobactin is synthesized and secreted by Escherichia coli. Its biosynthesis is performed by a series of enzymes encoded by the Ent gene cluster. Among the genes of this cluster, ybdB has not been implicated in enterobactin production to date. We demonstrate here an in vivo role for the hotdog protein EntH (YbdB) in the optimal production of enterobactin. Indeed, we showed that EntH is a thioesterase specifically produced under iron limitation conditions. Furthermore, EntH interacts specifically with the aryl carrier protein (ArCP) domain of EntB, a crucial bifunctional enzyme of the enterobactin biosynthesis pathway and a potential target of EntH thioesterase activity. A strain devoid of EntH is impaired for growth under iron limitation associated with the presence of the salicylate inhibitor, correlating with the diminution of enterobactin production under these conditions. Normal growth and enterobactin production are restored upon expression of entH in trans. Inversely, unnecessary overproduction of EntH provokes a fall of the quantity of siderophore produced under iron starvation conditions. Our findings point to a proofreading role for EntH during biosynthesis of enterobactin in vivo. EntH thioesterase activity could be required for cleaving wrongly charged molecules on the carrier protein EntB. This is the first description of such a role in the optimization of a nonribosomal biosynthesis pathway for a protein of the hotdog superfamily.

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“…1 C and D). Our toolkit consists of the broad-substrate-range enzymes Ptb-Buk (from C. acetobutylicum) (12), TesB (from E. coli) (13), and Pct (from Megasphaera elsdenii) (14). Herein we describe efforts to design, construct, and characterize the multifunctional pathway while exploiting the uniquely designed bypass strategy with the toolkit enzymes.…”

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confidence: 99%

Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways

Tseng¹,

Prather

2012

Proc. Natl. Acad. Sci. U.S.A.

104

110

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Microbial systems are being increasingly developed as production hosts for a wide variety of chemical compounds. Broader adoption of microbial synthesis is hampered by a limited number of high-yielding natural pathways for molecules with the desired physical properties, as well as the difficulty in functionally assembling complex biosynthetic pathways in heterologous hosts. Here, we address both of these challenges by reporting the adaptation of the butanol biosynthetic pathway for the synthesis of odd-chain molecules and the development of a complementary modular toolkit to facilitate pathway construction, characterization, and optimization in engineered Escherichia coli. The modular feature of our pathway enables multientry and multiexit biosynthesis of various odd-chain compounds at high efficiency. By varying combinations of the pathway and toolkit enzymes, we demonstrate controlled production of propionate, trans-2-pentenoate, valerate, and pentanol, compounds with applications that include biofuels, antibiotics, biopolymers, and aroma chemicals. Importantly, and in contrast to a previously used method to identify limitations in heterologous amorphadiene production, our bypass strategy was effective even without the presence of freely membrane-diffusible substrates. This approach should prove useful for optimization of other pathways that use CoA-derivatized intermediates, including fatty acid β-oxidation and the mevalonate pathway for isoprenoid synthesis.biochemicals | metabolic engineering | synthetic biology

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Thioesterase II of Escherichia coli Plays an Important Role in 3-Hydroxydecanoic Acid Production

Cited by 48 publications

References 34 publications

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

The Hotdog Thioesterase EntH (YbdB) Plays a Role In Vivo in Optimal Enterobactin Biosynthesis by Interacting with the ArCP Domain of EntB

Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways

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